3 Recombinant Gelatins

2) United States Patent Chang et al oy 0s) 03) wo ey @ os) @) (6) RECOMBINANT GELATINS Inventors: Robert C. Chang, Burlingame, CA. (US); Karl I. Kivieikko, Oulu (2) ‘Thomas B. Neff, Atherton, CA (US) David R. Olsen, Menlo Park, CA ( James W. Polarek, Sausalito, CA (US) Assignee: FibroGen, In., South San Franciseo, cAWS) Notice: Subject to any disclaimer, the tem of this patent is extended or adjusted under 38 USC. 154(b) by 0 days, Tis patent is subject to a terminal dis- citer: Appl. Now 11/139,377 Filed: May 27, 2008 Prior Publication Data US 200510229268.A1 Oct. 13, 2005 Related Continuation of application No. 091710239, filed oa Application Data Now. 10, 2000, now Pat. No. 6:992,172. Provisional application No. (204,437, filed on May 2000, provisional application No. 60/165,114, jon Nov. 12, 1999, 'US007393928B2 (10) Patent No.: US 7,393,928 B2 (45) Date of Patent: Jul. 1, 2008 G1) neu AGIK 38/17 (2006.01) Coon 300 (2006.01) (2) US.CL SAU/3S4;$300350; $36/28.1 -435/69.1; 439920.1; 435/325 (58) Fleld of Classification Search 530354, ‘30/380: $36/23.1; 435/320.1, 69.1, 328, ‘See aplication file for complete search history. 60) References Cited US. PATENT DOCUMENTS 5821.08 A + 101998. Gruskn eta. as714 6413742 BL* 72002 Olsen cal, #5001 Ga2897e BL* $2002 Olsen eal Bs 992.172 BL* 1/2005 Chang al 530364 (OTHER PUBLICATIONS Warten tal, Yeast 15, 1087-1096 (Aug 1999) * * cited by examiner Primary Examiner —Chik-Mi Kam (4) Attorney, Agent, or Firm—Jamnes B. Nesbitt on ABSTRACT Te present invention relates to recombinant gelatns and compositions thereof, and methods of prodcing and using the same, 24 Claims, 12 Drawing SheetsU.S. Patent Jul. 1, 2008 Sheet 1 of 12 US 7,393,928 B2 Figure 1 kDa 55 36 a1 14 1 2 3 4 Figure 3U.S. Patent Jul. 1, 2008 Sheet 2 of 12 US 7,393,928 B2 08 07-4 5 wean| 4 06 4 4 05 4 4 oD 044 4 03 4 4 02-4 4 o14 4 = 0.0 ; - VITROGEN BSA SEQID SEQID SEQID NO:18 NO:20 -NO:21 Figure 2A 14 foe le G25 MEAN] 4 104 — 7 08 4 4 oD 06 4 4 04 4 4 024 4 VITROGEN BSA SEQID SEQ ID SEQID SEQID 0-30 0-50 NO:19 NO:20 NO:21 NO:22 kDa kDa Figure 2BU.S. Patent Jul. 1, 2008 Sheet 3 of 12 US 7,393,928 B2 | 1 Figure 4A 123456789 10 ISOLATE NUMBER Figure 4B <10kDa 11 12 13 14 15 16 17 18 19 20 ISOLATE NUMBER Figure 5 GELATIN > 7.8 9 10 12345 6 FLOW THROUGH 0.5 M NaCl ELUATEU.S. Patent Jul. 1, 2008 Sheet 4 of 12 US 7,393,928 B2 kDa Figure 6A 31 A 241 << INTACT GELATIN 14 B <— PROTEOLYTIC FRAGMENT 6 kDa Figure 6B 31 21 ~< INTACT GELATIN 14 ~ PROTEOLYTIC o FRAGMENT kDa Figure 6C 31 21 14 ~< INTACT GELATIN ~< PROTEOLYTIC FRAGMENTUS 7,393,928 B2 Sheet 5 of 12 Jul. 1, 2008 US. Patent %0'% %0'L %S'0 wSz'0 | %SZ1'0 ‘%S290'0 %LE0O %SL0'0 %0'0 LACTALBUMIN Figure 7A WS %0'L %S'0 %S2'0 %SZ1'0 %$290'0 %LE0'0 %SL0'0 %0'0 SOYTONE Figure 7BUS 7,393,928 B2 Sheet 6 of 12 2008 Jul. 1 U.S, Patent eSINn 903 %1'0 ‘t-SINN 903 %2'0‘t-SINN ff NILW139 31NSdvO NILV139 VWOIS Ff ais MIN 8 Figure mod og SyW1 Lvad SIAVG H3NI31 ais MNUS 7,393,928 B2 Sheet 7 of 12 Jul. 1, 2008 U.S, Patent 4us'z ‘Hd 4uz ‘ed sus't ‘eHd 4ut ‘Hd sys‘0 ‘Hd 440 ‘EHO als MW Jug'2 ‘Hd Juz ‘2d jus’ ‘zHd JUL ‘Hd sus'o ‘zd 4u0 ‘ZHd GLs MW Figure 10B Figure 10A dupe ‘SHO 440g ‘SHA sug ‘SHd Jygh ‘SHA Jupl ‘sHd 4yo ‘SHd ais MW supe ‘pHd suog ‘bHd 4ugL ‘pHd 4Ug'LL “PHO 4us ‘pHO 4uy ‘vHd 240 ‘HO ais MW Figure 10D Figure 10CUS 7,393,928 B2 Sheet 8 of 12 Jul. 1, 2008 U.S, Patent supe ‘LH 4uez ‘LHd s4oz ‘ZHd duet ‘ZHd tug ‘ZH 4ubL ‘ZHd 440 'ZHd J ails MW 4ube ‘SHO suze ‘SHO 4402 ‘SHd Jul ‘SHO F 4491 ‘SHO sup ‘SHO 2u0 ‘9Hd ais MW Figure 10F Figure 10E syok ‘sud tug ‘SHd 4yg ‘SHd up ‘SHO § sug ‘SH 440 ‘sHd CLS MW & 4u0L ‘pHd 24g ‘yHd 1ug ‘bd 4up ‘pH 4uz ‘PHO 240 'bHO als MW Figure 11B Figure 11AUS 7,393,928 B2 Sheet 9 of 12 Jul. 1, 2008 U.S, Patent syoL ‘ZHd Jug ‘2Hd § 4p ‘ZH 4Uz ‘LHd 4yo ‘2ZHd ais MN 440} ‘gHd dys ‘9Hd aug ‘9Hd 2Up ‘SHA 1uz ‘9Hd § 440 ‘9Hd aLs MW Figure 11D Figure 11C jug ‘ZHd 4Up ‘ZH 4Uz ‘ZH (11DuebeII00-4s 4yg ‘LH 4Uy “LH 4uz ‘LH (1uebe}}oo-ys ais MW sue ‘2Hd 4uz ‘2H JUL ‘ZH 4us'0 ‘ZHd 4yo ‘zHd jug ‘ZHd sug ‘ZHd dup ‘ZHd tue 'LHd ais MW th-collagen TYPE III th-collagen TYPE I Figure 12B Figure 12AUS 7,393,928 B2 Sheet 10 of 12 Jul. 1, 2008 U.S. Patent JUEZ “X ESBAIOG 4ug “x esBeIod Jug ‘x esPelold JUp "X OSBOIOId Juz ‘X @SBaIOd dug ‘ureded & lug ‘uredeg uy ‘uledeg Juz ‘ueded F GISMW F 105KD 53KD 34KD 23KD 13 Figure 13KD 7KD 4KD 8% Tris-Glycine €€:0N GI 03S 22:ON al 03S 42:0N I 03S 02:0N al OAS 6LON GI 03S 8LON GI 03S thel M Figure 14 M rhel rholll vitrU.S. Patent Jul. 1, 2008 Sheet 11 of 12 US 7,393,928 B2 Figure 15A at(I) a1(1) @2(1) @2(1) +CNBr +CNBr FULL LENGTH a2(I) FRAGMENT OF o2(1) Figure 15B thel cel(I) _o1(l) o2(1) _02(l +CNBr +CNBrU.S. Patent Jul. 1, 2008 Sheet 12 of 12 US 7,393,928 B2 —s— thel —*&— SEQ ID NO:18 —*— SEQ IDNO:19 —#®— SEQIDNO:20 Coma tt ere —+— SEQ ID NO:22 25 4 1 —— __ SEQ IDNO:33 oD 204 4 0.0 T T T T T T 10:62 3.93) 1111. 087120123). 0.0411= 0.013 Cone. (ug) Figure 16US 7,393,928 B2 1 RECOMBINANT GELATINS ‘This application isa continuation of US. application Ser. No. 09/710,238, filed on 10 Nov. 2000, now U.S. Pat No. 6,992,172, which claims the benefit of US. Provisional Application Nos. 601204437, filed 1S May 2000, and 60/165, 114, filed 12 Nov. 1999, the specifications of which are incorporated herein by reference in ther entireties. FIELD OF THE INVENTION This invention relates to recombinant gelatns nd to compositions and agents comprising recombinant gelatins, 10 methods of producing recombinant gelatins, and 1 the vse of these gelatins in various applications BACKGROUND OF THE INVENTION Gelatinisa derivative of collagen, a prinepalstrutura and ‘connective protein in animals. Gelatin is derived from dena ‘uration of collagen and contains polypeptide sequences av- ing Gly-X-Y repeats, where X and Y are most often proline and hydroxyprofine residues. These sequences contribute 0 teple helical structure and afect he gelling ability of gelatin polypeptides. Currently available gelatin is extracted through processing of animal hides and bones, typically from bovine find porcine sources. The biophysical properties of gelatin make ita versatile material, widely used ina variety of appli ‘ctionsand industries, Glatinis sed forexomple, in nomer- ‘ous plamaceutical and medieal, photographie, industeal, ‘cosmetic, and food and beverage products and processes of ‘manufacture. Gelatin is thus & commercially valuable and versatile product Manufacture of Gelatin Gelatnis typically manufactured from naturally occurring collagen in bovine and porcine sourees, in particule, from hides and bones. In some instances, gelatin can be extracted from, for example, piscine, chicken, or equine sources, Rew materials of typial gelatin production, sich as bovine hides ‘and bones, originate from animals subject to goverament- certified inspection and passed fit for human consumption, There is coneem over the infectivity ofthis rae materia, dve to the presence of contaminating agents such as transmissible spongiform encephalopathies (TSEs), particularly bovine spongiform encephalopathy (BSE), and serapie, etc. (See eg, Rohwer, RG. (1996), Dev Biol Stand 88:247-256,) Such issues are especialy eitel to gelatin used a pharma- ‘ceutical and medial applications Recently, concer about the safety of these materials, 3 significant portion of which ae derived fom hovine sources, has increased, causing various gelatin-containing products to become the focus of several regulatory measures o reduce the potential risk of transmission of bovine spongiform encepha- Fopathy (BSE), linked to new variant Crenzfeld-Jakob dis- ‘ease (aC ID), fatal neurological disease inhhumans. Thereis, ‘concern that purification steps curently used in the process ing of extracting gelatin from animal tissues and bones may not be sufficient to remove the likelihood of infectivity dve to SE-carying tissve (ie, brain tissue, ee.) USS. and Buropean manufacturers specify that raw material ‘or gelatin to be included in anand or human food produets or in pharmaceutical, medical, or cosmetic applications must not be abisined from a growing number of BSE countries, In dition, regulations specify that certain materials, eg. bovine brain tssne, are not use inthe production of gelatin (Current production processes involve several purification ‘and cleansing steps, and ean require harsh and lengthy modes, 0 o 2 of extraction, The animal hides and bones are treated rendering process, and the extracted material i subjected to various chemical teatment, including proloaged exposure to highly aeidie or alkaline sokutions. Numerous purification steps can involve washing and filtration and various heat treatments. Acid demineralization and lime treatments are ed to remove impurities such as non-cllagenous proteins [ones must be depressed. Additional washing and filtration steps, jon exchanges, and other chemical and sterilizing ret- scare added to the proces a further purify the material. Furlermore, contaminants and impurities can sil remain aller processing. and the resultant gelatin product must Us ‘ypically be clarified, purified, and often farther concentrated before being ready for use ‘Commercial gelatin is generally classified as type ortype 1B. These classifications reflect the pre-realment extraction sources receive as part ofthe extraction process. Type A is generally derived from acid-processed materials, usually or- eine hides, and type B is generally derived from afkaline- or Jime-processed materials, usually bovine bones (ossein) and hides TInextracting ype A gelatin, the press generally involves subjecting fresh or frozen porcine bides to successive wash- ings with water and treatments with dilute acids. The aeid- treated skins are washed again and are then subject 10 repeated extraction steps in which they are trated with hot ‘water, partially hydrolyzing the collagen present. The result antextracts, dilute solutions of peat, ae tered and evapo rated, and the resultant concentrates are allowed to cool oF chilled to a gel. The gel is subsoquently treated in drying tunnels, or by continuous dryers or aher drying devices. In the Fimed process, type B gelatin i derived from donor hides and skin trimmings washed and then treated with lime The Hime treatment ean take as long as from one to three sonths, dis usually around sixty days, The lied hides are ‘washed and treated with dilute acs, The hides are thea Fyrolyzed with hot water and te resulting extracts ae pro- cessod as deseribod above for the aeid-testment process ‘Type ft gelatin can also be processed from osscin sources, The hard bones are washed, degreased, anc leached with stccessive treatments of dilste acids, such as hydrochloric acid. The acid treatment reacts with the mineral contents of bone, which are removed along with the acidic solution leaving ossein, or demineralized bones. This organic bone ste, washed fre of residual avid, i dred for storage or ‘immediately Timed. After Timing, ossein is subsequent)y treated as described above forthe produetion of gelatin from bovine hides, In all ease, after final fering, demineralization, concentration, and drying steps, the resultant gelatin product is divided ino batches, subjected to various physical, chemical, and bacteriological tests to detemnine grade and purity, and ground and blended according to commercial ‘eirements ln both type A and B extraction processes, the resultant gelatin prodict typically comprises a mixture of gelatin molecules, in sizes of from a few thousand up to Several hundred thousand Daltons. Fish gelatin, classified as yelling or non-gelling types and ‘ypieally processed as Type A gelatin, is also used in certain commercial applications. Gelling types are usually derived from theskinsof warm water ish, whilenon-gelling types are typically derived from cold water fish. Fish gelatine have ‘widely varying amino acid compositions, and differ from ‘animal geatns in baving typically ower proportions of pro- Tine and hydroxyproline residues. In contrast to animal gelatins, fsh gelatins typically remain Tguid at much lower temperature, even at comparable average molecular weighs. As ‘With other animal gelatin, fish gelatin i extracted by reaUS 7,393,928 B2 3 ment and subsequent hydrolyzation of fish skin. Again, as ‘with animal extraction processes, the process of exacting fish yelatn results ina produet that lacks homogeneity, SUMMARY latin is an essential prove used in wide-ranging ay ‘ations. The diverse uses of gelatin rely on diferent charac teristics and properties of this ubiquitous mixture of proteins. ‘Current methods of extraction resilt n gelatin produc hat ‘a heterogencos mixture of proteins, containing polypeptides with molecular weight distributions of varyingranges. It is sometimes necessary to blend various fots of product in ‘onde o obisin a gelatin mixture with the physical properties appropriate for use in a desired application. ‘A more homogeneous product, and one produced by more reproducible means, would be desirable. The availability of @ homogeneous material with reproducible physical character istics would be desinble, for example, in various products and processes, where the availabilty of peat cchanicteristics, such asa fixed range of molecular weight would allow fora reproducible and contolled performance ‘There is thus a need fora reliable and reproducible means of gelatin production that provides a homogeneous prodt with ‘controlled characteristics, Inaddition, inthe pharmaceutical, cosmetic, and food and beverage industries, especially: there isa need fora source of pelatin other than that obtained through extraction from ani ral sources, e., bovine and porcine bones and tissues. Fur- ther, as currently available gelatin is manufetured fom ani mal sources such as bones and tissues, there are concerts relating othe undesirable immunogenicity and infectivity of pelain-contzining products. (See, ex, Sakaguchi, Me al. (1999) 3. Aller-Clin. Immunol. 104:695-699; Miyazawa etal. (1999) Vaeine 17:2176-2180; Sakaguehi etal (1999) Immu- nology 96:286-290; Kelso (1999) J Aller. Clin Immunol. 103:200-202; Asher (1999) Dev Biol Stand 99:41-44; and Verdrager (1999) Lancet 354:1304-1305,) In audition, the availability of a substture material that does not undergo ‘extraction from animal sources, e., tissues and bones, will, address various ethical, religious, and social dictates. recombinant moterial that does not require extraction from ‘animal Sources, such a tssues and bones, could be used, for ‘example, in the manufacture of foods and other ingested products, including encapsulated medicines, that are appropriate fr use by people with dietary restrictions, for example, those who follow Kosher and Hala aw ‘While gelatin producers and end-users have searched for and tested a number of natural and synthetic substitutes for ‘heanimal-source gelatin currently available, universal substitute has not yet boon found, Alkeratives have been identi fed for a few applications, such as the use of cellulosic ew materials in VCAPS capsules (CAPSUGEL; Mortis Pl N3J.),or the proposed use of non-natural wlatn-ike proteins from mouse and rat collagen sequences in photographic ‘emulsions. (See, eg, Werten, M. W. et a. (1999) Yeast 15:1087-1096; and De Wolf, Anton etal, Buropean Appl tion No, EPIOI4176A2,) However, for most geltin-based processes and products, the performance characteristics of this key material have not been duplicated, aad substiutes have not been adopted. Thus, there isa need for a means of producing gelatin in a synthetic and reproducible manner ‘wherein theresullant product ean serveas a rational substitute with the desired performance characteristics, Tn summary, there is 9 need for @ universal replacement material that can provide performance characteristics of gela- While allowing for a more reproducible and controlled Specific x 4 source of product. There isa need for methods of producing alain that do not require harsh and lengthy processing, and or methods of manufacturing gelatin that result in a more ‘unifoem product and that are capable of stably producing significant amounts an different types of gelatin appropriate ordiverse applications. There isa ned fora versale gelatin product that is readily adaptable for diferent uses and that answers existing health and other concerns, Te present invention solves these and other needs by providing a universal replacement material, obtained recombinant, appropriate for use in the extraordinarily diverse spectrum of applications in which gelatin is currently used ‘The present materials can be designed to possess the proper- ‘ies and characterises desired for particular applications, and can this provide new properties and uses previously ‘unavailable, SUMMARY OF THE INVENTION The present invention is directed o recombinant gels to compositions and agents comprising recombinaat gelatin, and to methods of producing and using recombinant geatns. Tone aspect, the present invention prides a composition comprising recombinant gelstin. In one embodiment, the ‘recombinant gelatin as a molecular weight selected fom the soup consisting of about § kDa, 8 kDa, 9 kDa, 14 kDa, 16 kDa, 22kDa, 23 kDa, 36kDa, 44 kDa, and 65 kDa. Inanother ‘embodiment, the recombinant gelatin has a molecular weight ‘ange selected from the group consisting of about to SOKDa, bout 10 1930 kDa, about 30 t 50 kDa, about 10t0 70 kDa, fahout SOKDa to TOKDu about SO 100 kDa, about 100 150 kDa, about 150 to 200 kDa, about 200 to 250 kDa, about 250 {© 300 kDa, and about 300 19 350 kDa. In one aspect the recombinant gelatin has a molecular weight greater than 300 kDe, In another aspect, the invention encompasses a recombi ‘sant gelatin having Bloom strength selected from the group consisting of $0, 100, 150, 200, 250, and 300. In further embodiment, the Bloom strength is between 0 and 100. In certain embodiments the present invention provides a ‘composition comprising recombinant gelatin wherein the recombinant gelatin non-hydroxylated, fully hydroxylated, ‘orpactilly hydroxylated. In various aspects, the recombinant gelatin has a percentage hydroxylation selected from the 0p consisting of 20 to 80%, 30 to 80%, 40 0 80%, 60 40 80%, 2010 60%, 3010 60%, 4010 60%, 2010 30%, 2010 40%, ‘and 3010 40%. ia other embodiment, the recombinant gel ‘in is fully hydrolyzed, partially hydrolyzed, or non-hydeo- Ize. Tnone aspect, the present invention provides composition comprising recombinant gelatin, wherein the recombinant aelatin comprises « homogeneous mixture of recombinay gelatin polypeptides. Inanothor aspect, the recombinant gela- ‘in comprises a heterogeneous mixture of recombinzant gel tin polypeptides none embodiment, the present invention provides acom- positon comprising recombinant gelatin wherein the recombinant gelatin is derive from one type of collagen free of any other collagen. In particular embodiments, the one type of collagen is selecie from the group consisting of type Is type Type ll ype IV, ype ¥, type VI, type VIL, ype VIM, pe IX, type X, type XI, type Xl, type Xl, type XIV, type XV, type XVI, ype XVI, type XVII, type XIX, and type XX collagen. Compositions of recombinant gelatin wherein the recombinant gelatin hs endotoxin levels of below 1.000 [ELlimg, below 0.500 Fig, blows 0.050 Fig. and below 0.005 U/mg are contemplated.US 7,393,928 B2 5 In specific embodiments, the recombinant gelatin of the presen invention comprises an amino acd sequence selected Jom the group consisting of SEQ ID NOs:15, 16,17, 18, 19, 20,21, 22, 23, 24,25, 30,31, and 33. Polymucleotides encode ing these amino acid sequences are also provided, as are ‘expression vectors and fost cells containing the polynvcle- ‘tides, In certain aspects, the hos cells of the presen inwen~ tion are prokaryotic or eukaryotic. In one embodiment, & ‘eukaryoti host cells selected from the group consisting of 3 yeast cell, an animal cell, an insect cell, a plant eel, and 2 JTungal cel. The present invention further provides transgenic ‘animals and (rinsgenie plants comprising the polynuele ‘tides. Recombinant gelatins compesing. an_amino acid Sequence selected from the group consisting ofSEQ ID NOs: 26, 27, 28, and 29 are also provided. Tone aspect, the present invention encompasses methods ‘of producing the recombinant gelatins, One method comprises providing recombinant collagen or procollagen or lrag- ments or variants thereo!; and processing the recombinant ‘collagen or procollagen or Fragments oF variants thereof to produce recombinant gelatin. In one aspect, the recombinant Collagen processed 10 recombinant gelatin is recombinant human collagen. Ina further aspect, she recombinant collagen is produced by co-expressing at least one polynucleotide ‘encoding a collagen or procollagen and at least one poly neleotide encoding a collagen post-translational enzyme or subunit thereof. In certain embodiment, the post-iranslational enzyme is proly! hydroxylase Tn another method according t0 the present invention, recombinant gelatin is produced diretly from an altered collagen construct. Ina further embodiment, the recombinant eatin is produced by co-expressing the altered collagen ‘constrict and at least one polynucleotide encoding a post ttanslational enzyme or subunit thereof. In one embodimen the post-ranslational enzyme is prolyl hyeroxylase ‘Methods of producing recombinant gelatins having selected melting temperatures are also provided. In one ‘embodiment, the method comprises conferring on the recombinant gelatin percentage hydroxylation that corresponds to the selected melting temperature, In a further embodiment, the conferring step comprises producing recombinant gelatin from an altered collagen construc in the presence of prolyl hydroxylase. ln other aspects, the conferring step comprises “driving recombinant pelatin from hydroxylated recombinant collagen, or comprises. ydroxylating noa-hydroxylated recombinant gelatin ‘Various uses of the recombinant geatins of the present ‘invention are contemplated. In particular, the present invention comprises encapsulans, stabilizing agents, film-forming agents, moisturizing agents, emulsifiers, thickening agents, zelling agents, colloidal agents, adhesive agents, foceulating gents, and refining agents comprising rocombinan gelatin, “The present invention provides in one embadimment «phar ‘maceutical eomposition comprising recombinant gelatin. In Trter embodiment, the recombinant gelatin is human recombinant gelatin, In another embodiment, the recombinant gelatin is non-immunogenic. In specific embodiments the present invention provides a hard gel capsule a soft gel ‘capsule, a tablet coating, a plasma expander, a colloidal vol- ‘ume replacement material, graf coating, medical sponge, ‘a medical plug a pharmaceutical stabilizer, and a mierocar- Fier comprising. recombinant gelatin, In one aspect, the present invention encompasses a kit comprising a composi tion comprising recombinant gelatin, and a device for doliv- ‘ering the composition 1o a subject. "An edible composition comprising combinant gelatin is alsocontemplated, as.are protein supplements, ft substitutes, 0 o 6 svtional supplements, edible coatings, and various ‘icroencapsulants comprising recombinant gelatin, Photo- raphic compositions comprising recombinant gelatin are also contemplated, as are embodiments in which recombinant gelatin is partially o filly hycroxylated, The invention fur ther provides cosmetic composition comprising recombi- ant gelatin Ta other embodiments, the invention encompasses a cos: ‘metic composition comprising recombinant gelatin, an indy ‘cal composition comprising recombinant gelatin, a cell cle ‘ure composition comprising recombinant gelatin, and a ‘composition for laboratory use comprising recombinant gelatin. Further embodiments, such as miemarray’s comprising the recombinant gelatins of the present invention oF poly- uceotides encoding these recombinant gelatins, are contemplated. [BRIEF DESCRIPTION OF THE DRAWINGS. FIG. 1 sets forth results showing the expression of recom binant gelatin. FIGS, 2A and 2B set forth results demonstrating that recombinant gelatin support cell attachment FIG. 3 sets forth results demonstrating the production of proteolytically stable recombinant latins, FIGS, 4 and 4B set fort results demonsteating the peo- duction of hydroxylated recombinant gelatins FIG. 5 sets forth results showing the purification of recom. binant gelatin following in vitro hydroxylation, FIGS. 6A, 68, and 6¢ sot fort results showing te stability ‘of recombinant gelatns expressed in the presence or absence of prolyl 4-hydroxyiase. IGS, 74 and 72 set forth results demonstrating enhanced recombinant gelatin expression by supplementation of expression media FIG. 8 ses forth results comparing commercially available aelatins to cross-linked recombinant gelati FIG. 9 sets forth results comparing the molecular weight tural or functional characteristic of collagen for example, (Giy-X-Y), domain, The tent “procollagen” refers to a procollagen corre sponding to any one ofthe collagen types I through XX, as ‘wellas toa procollagen coresponding any other collagens, ‘whether natural, synthetic, somi-synthotc, or recombinant, that possesses ‘additional C-terminal and/or N-terminal propeptides of telopeptides that assist in trimer assembly, Solubility purifieaion, orany other function, and that then are 0 o 8 subsequently cleaved by N-proteinase, C-proteinase, or other enzymes, eg., protelstic enzymes associated with collagen production. The tem procollagen specifically encompasses variants and fragments thereof, and functional equivalents and derivatives thereof, whieh preferably retain at least one Structural of functional characteristic of collagen, for example, (Gly-X-Y), domi, “Gelatin” as used herein refers to any gelatin, whether exttacied by traditional methods or recombinant oe biosynthetic in origin, or to any molecule having a least one src= tural andor funetional characterise of gelatin, Gelatin is ‘currently obtained by exiscton from collagen derived from animal (eg, bovine, porcine, chicken, equine, piscine) sources, eg, bones and tissues, The term gelatin encompasses both the composition of more than one polypeptide included in a gelatin product, as well as an individval polypeptice contributing to the gelatin material, Thus, the fern recombinaat gelatin as used in relerence tothe present invention encompasses both a recombinant gelatin material comprising the present gelatin polypeptides, as well as an individual gelatin polypeptide ofthe present invention Polypeptides from which gelatin can be derived are polypeptices such as collagens, procollagens, and other polypeptides having atleast one truetural andor functional characteristic of collagen. Such polypeptide could include a single collagen chain, ora collagen homotrimer of heteroti- ‘mer, or any fragments, derivatives, oligormers, polymers, oF subunits thereo containing atleast one collagenous domain (aGly-X-Y region). Theterm specifically contemplates engi- ered sequences not found in nature, such asalterel collagen constnicts, et. An altered collagen construct isa polynicle- ‘tide comprising sequence that isaltered through deletions, additions, substitutions, or other changes, from a naturally ‘curing collagen gene. ‘An “adjuvant” is any agent added toa drug or vaccine to increase, improve, oF otherwise aid its elfet. An adjuvant ‘sed in a vaceine formulation might be an immunological ‘agent that improves the immune response by producing a ‘non-specific stimulator of the immune response. Adjuvants fare often used in non-living vaccines, “The temns “allele” or “allelic sequence” refer to alternative forms of genetic sequences, Alleles may result fom atleast ‘one mutation in dhe melee acid sequence and may rest ia altered mRNAs or polypeptides whose structure or function ‘may or may not be altered. Any given natural or recombinant gene may have none, one, of many allele forms. Common ‘mutational changes Which give rise to alleles are generally ascribed to natural deletions, additions, oF substitutions of neleotides. Fach ofthese types of changes may occur alone ‘or in combination with the others, one oF mote times in @ sven sequence “Alter polynucleotide sequences include those with deletions, insertions, or substituions of different nucleotides resulting in polymicleoide that encodes the same ora fune- tionally equivalent polypeptide. Included within this defin- tion are sequences displaying polymomphisms that may or ‘may not he readily deteciale using particular oligonile- ‘tide probes or through deletion of improper or unexpected Liybrdization to alleles, with locus othr than the normal ‘chromosomal oeus fr the subject potynucleotide sequence. “Altered” polypeptides may contain deletions, insertions, ‘orsubsttuions of aminoacid resides which produce silent ‘change and result in a finctionally equivalent polypeptide Deliberate amino acid subsittions may be madéon the basis of similarity in polarity, charge, solubility, hydrophobicity bbydrophilicity. andor the amphipathic nature of the reicucs as long as the biological or immunological activity of theUS 7,393,928 B2 9 ‘encoded polypertide is retained. For example, negatively ‘charged amino acids may include aspartic acid and glutamic acid; positively charged amino aids may include Iysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values may inelnde leucine, ‘solevcine, and valine, alyeine and alanine, asparagine and lutamine, serine and threonine, and phenylalanine and "Amino acid” oF “polypeptide” sequences or “polypep= tides." as these terms are used herein, refer to oligopeptide peptide, polypeptide, or protein sequences, and fragments thereof, and (© natally occurring or synthetic molecules. Polypeptide or amino acid fragments are any portion of & polypeptide which retain atleast one strcturl andr funetional characteristic of the polypeptide. In at least one nbowtiment ofthe present invention, polypeptide fragments ae those retaining atleast one (Gly-X-Y), region. Te term “animal” asitis used in reference, for example, to “animal collagens” encompasses any collagens, derived om ‘animal sources, whether natura symthesic, semi-synthetic, oF recombinant. Animal sources include, for example, mamm Tian sources, including, but not limited to, bovine, porcine. and ovine sources, and other animal sources, including, but not limited to, chicken and piseine, equine, dent, and n0n- vertebrate sources. Antigeniety” relates to the ability ofa substance to, whe introduced into the body stimulate the immune response and the production of an antibody. An agent displaying the prop- ‘erty of antigenicity is referred toas being antigenic. Antigenic ‘agents can include, bt ate not Fimited to, a variety of macro= molecules such as, for example, proteins, lipoproteins polysaccharides, neleie acids, bacteria and bacterial compo- ents, and viruses and viral components The terms “complementary” or “complementarity,” as used herein, referto the natural binding of polynucleotides by base-pairing, Forexample the sequence“A-G-1” binds tothe ‘complementary sequence “I-C-A.” Complementarity between two single-stranded molecuies may be “partial,” shen only same of the nucleic acids bind, oF may be com= plete, when total complementarity exists betwen the single ‘mand molecules, The degree of complementarity between rucleic acid strands has significant effets on the efficiency tnd strength of hybridization between nucleic acid strands ‘This is of particular importance in amplification eaetions, Which depend upon binding between nucleic acids strands and inthe design and use, for example, of peptide nveleie acid (PNA) molecules. A “deletion” is @ change in an amino acid or nucleotide sequence that results in theabsence of one or more amino acid residues or nucleotides The term*derivative""3s applied to polynucleotides, refers 1 the chemical modification ofa polynucleotide encoding & particular polypeptide or complementary to a polynucleotide ‘encoding “particular polypeptide. Such modifications include, for example, replacement of hydrogen by an alkyl cyl or amino group. As used herein to refer to polypeptides, the term “derivative” refers toa polypeptide which is modi fied, for example, by hydroxylation, glycosylation, pegyla- tion, orby any similar process. The tem “derivatives” enc passes those molecules containing at least one structural and! ‘r Tunetional characteristic ofthe molecule feom which tis derived ‘A molecule issaid to hea “chemical derivative” of another ‘molecule when it contains additional chemical moisties not ronmally part ofthe molecule. Such moieties can improve themolecule’s solubility, absorption, biological halflife, and the lke, The moieties can alternatively decrease the toxicity 0 o 10 of the molecule, eliminate or attenuate any undesirable side effect of the molecule, and the like. Moictes capable of mediating such effets are generally available inthe art and can be found for example, in Remington's Pharmaceutical Sciences, supra, Procedures for coupling such moieties 10 a smolecleare well known i the art ‘An “excipient” as the term is used herein is any ineet substance used asa diluent or vehicle in the formulation of a ddnig, a vaceine, or other pharmaceutical composition, in ‘order to confer a suitable consisteney or form to the dug, vaccine, or pharmaceutical composition ‘The tenn “funetional equivalent” as itis used herein refers 1 polypeptide or polyauelootie that possesses atleast one fmetional ‘andor structural characteristic of particular polypeptide or polymicleotide. functional equivalent may feantain modifications that enable the perfomance ofa spe- cif finetion. The term “functional equivalents intended to include fragments, mutants, hybrids, variants, analogs, or chemical derivatives of s molecule ‘A “fasion protein” sa protein ia which peptide soquences from different proteins are operably likes. ‘The term “hybridization” refers fo the process by whieh a nucle acid sequence binds to # complementary sequence ‘ough bate pairing. Hybridizaion conditions can be defined by; for example, the concentrations of salt or forma- snide in the prchybridization and hybridization solutions, oF by the hybridization temperature, and are well known in the an. Hybridization can oceur under eontionsof variousstin- gency. In particular, stringency can be increased by reducing the ‘concentration of sl, inereasng the concentration of forma mide, or raising the hybridization temperature. For example {or purposes of the present invention, hybridization under high stringency conditions occurs in ahout 0% formamide at about 37°C. to 42°C, and under reduced stringeney condi ‘ions in about 35% to 38% formamide at about 30° C. to 35° CInpanticular, hybridization occurs in conditions of highest stringency at 42°C in SOM formamide, Sx8SPE, 0.3% SDS, ‘nd 200 jigiml sheared snd denatured salmon sperm DNA. ‘The temperature range corresponding t a particular level of singeacy ean be further narrowed by methods known ia the art, For example, by calculating the purine to pyrimidine ratio ofthe mclet aid interest and adjusting the tempera ‘ure accordingly. To remove nonspecific signa, blots can be sequentially washed, for example, at room temperature under increasingly stringent conditions of up to 0.1xSSC and 0.5% SSDS. Variations on the above ranges and conditions are well -nowa in the ar. “Immunogenicity” relates 10 the ability 10 evoke an ‘immune response within an organism. An agent displaying the property of immunogenicity is referred toas being immu rnogenic, Agents can include, but are not listed wo, a variety ‘of macromolecules such as, for example, proteins, lipopeo- teins, polysaccharides, nucleic acid, bacteria and bacterial components, and vies and viral components, Immuno- genie agents offen havea fairly igh molecular weight (us- ally greater than 10 kD). infectivity” refers 10 the ability to be infective or the ability to produce infection, refering to the invasion and ‘multiplication of mieroorganisms, such as bacteria or viruses ‘within the body. “The teams “insertion” or “addition” refer to a change in a polypeptide or polynscleoide sequence resulting in the add tion of one or more amino acid residues or nucleotides, respectively, as compared to the naturally occuring mol- eau.US 7,393,928 B2 u The term “isolated” as used herein refers to @ molecule separated ot only from proteins, et. that are presen in the natural source ofthe protein, butalso Irom other components jin general, and preferably refers to a molecule found in the presence of, ifanything, only a solvent, butler, ion, or ather ‘component aoemally present in a solution ofthe same. As twsed herein, the terms “isolated” and “purified” do not ‘encompass molecules preset in their natural source. The term “microarray” refers to any arrangement of nucleic acids, amino acids, antibodies, etc, om a substrate ‘The substrate can be any suitable support, e., beads, gla, paper, nitrocellulose, nylon, or any appropriate membrane ‘etc. substrate can be any rigid or semi-rigid support includ Jing, but not limited to, membeanes, filters, wafers, chips slides, fibers, beads, inchading magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles, capil- Jaries, ete. The substrate can provide a surface for costing and/or can havea variety of surlace forms, suc as wells, pins, twonches, channels, and pores, 19 which the nucleic acids, amino acids, et, may be bound ‘The term “microorganism” can include, bt isnot limited to, viruses, bacteria, Chlamydia, rieketsias, mycoplasmas tureaplasmas, fungi, and parasites, including infectious pars sites such as protozoans. The terms "nucleic cid" or “polynucleotide” sequences oF “polynucleotides” refer to oljgonuceotides, nucleotides, or polynucleotides, or any fragments thereof, and to DNA of RNA of natural or synthetic origin which may be single- or double-stranded and may represent the sense of antisense strand, to peptide nucleic acid (PNA), or to-any DNA-like or RNA-like material natural or synthetic in origin, Polynucle- ‘lide frogments are any portion ofa polynucleotide sequence that retains atleast one stctural or fuctional characteristic ‘of the polynveleatide, In one embodiment of the present Jnventio, polynucleotide fragments ae those that encode at least one (Giy-X-Y), region, Polynucleote fragmenis can beof varible length, forexample, greater than 60 nucleotides in Tength, a least 100 nucleotides in length, at Teast 1000 nucleotides in length, ot least 10,000 nucleotides in length Te phrase “percent similarity” (% similaity refers tothe percentage of sequence similarity found in a comparison of| ‘wo or more polypeptide or polynucleotide sequences. Per- ‘ent similarity canbe determined by methods well-known in the an. For example, percent simularity between amino acid sequences can be calculated using the clustal method. (See ‘eg, Higgins, D. G. and P. M. Sharp (1988) Gene 73:287~ 24) The algorithm groups sequences into clusters by exam- ‘ning the distances between al pairs, The clusters are aligned pairwise and then in groups. The percentage similarity between fo amino acid sequences, eg, sequence A and sequence B, is calculated by dividing the length of sequence AAvminos the numberof exp residues in sequence A, minus the ruumber of gap residues in sqquence B, ino the sum of the reside matehes between sequence A and sequence B, times ‘onehundred. Gapsoflow or of ao homology between the two famino acid saquences are not included in determining per ‘centage similarity. Percent similarity can he calculated by ‘other methods known in the art, for example, by varying, using programs such asthe MEGALIGN proggam (DNAS- TAR Inc., Madison, Wis), As used herin, the term “plant inchudes reference to one ‘or more plants, Ze, any eukaryotic autotrophic organisms such as angiosperms and gymnosperms, monotyledns and icotyledons, ineluding, bul not limited to, soybean, come, alfalfa, Rox, tomato, sugar, beet, sunflower, potato, tobacco. maize, wheat, rice, lettuce, banana, cassava, salllower, oil 0 o 12 seed, rape, mustard, canola, hemp, algae, kelp, ete. The ter “plant” also encompasses one or more plant cells. The term “plant cells” includes, but is aot imited to, vegetative iss and organs such as soeds, suspension cultures, embryos, mer- jstematie regions, callus tisste, leaves, roots, shoots, game- tophytes, sporophites, pollen, tubers, corms, bulls, flaws, fiuits, cones, microspores, ee. ‘The term” post-ranslational enzyme" rofersto any'enzymne ‘hat catalyzes post-translatonal modification, for example, any collagen or procollagen. The term encompasses, but is ‘not limited to, for example, prolyl hydroxylase, peptidy pro- Jl isomerase, collagen galactesy! hydroxylysyl glucosyl transferase, hydroxylysyl galactosy! transferase, C-proein- ase, N-proteinase, Iysyl hydroxylase, and ysyl oxidase As used herein the term “promoter” generally refers 03 regulatory region of nucleic acid sequence capable o initiat- ing. directing, and mediating the transcription of polymucle- otk sequence, Promoters may additionally comprise reog- fon sequences, such as upstream or downstream promoter elements, which may influence te transcription rate. ‘The term “non-consittive promoters” refers to promoters that induce transcription via 2 specific tissue, oF may be clenwise under environmental or developmental controls, ‘and includes represtble and inxlucile promoters sel as tissue-prefered, tissue-specific, and cell type-specific pro- ‘moters. Sueh promoters include, but are not Iited (0, the Adil promoter, inducible by hypoxia or cold stress. the isp70 promoter, inducible By heat stress, and the PPDX ranote,induible by light, Promoters which ae “tisue-prefered” are promoters tht preferentially initiate transcription in certain tissues. Prom ers whieh are “tissue-specific” are promoters tht initiate transcription ony in certain issues. “Cell type-spocific®pro- rmoters are promoters which primarily drive expression in certain cell ypes in at least one organ, for example, vascular alls Inducible” or “repressible” promoters are those under control of the environment, such tat transcriptions effected, orexample, by an environmental condition schas anaerobic ‘conkltions, the presence of light, biotie stesses, ete, or in response 10 internal, chemical, or biological signals, e alyeeraldehyde phosphste dehydrogenase, AOX! and AOX2 ‘methanol-inducible promoters, oF to physical damage. As used herein, the tern “constitutive promoters” refers to promoters tat initiate, diet, oF mediate tanseription, ad fre active under most environmental conditions and states of ‘development of cell differentiation, Examples of constitutive promoters, include, but are not limited to, the cauliflower ‘ost virus (CaM¥) 35, the '- 0 2-promoter derived rom TTDNA of Agrohacteriuam tumefaciens, the ubiquitin 1 pro- voter, the Saas promoter, the einsamy! sleohol dehydro ge- nase promoter glyveraldchyde debydrogenase promoter ad the Nos promote, et. ‘Te term “purified” at it is used herein denotes thatthe indicated molecule is present in the substantial absence of other biological macromolecules, eg, polynucleotides. pro- ‘eins, and the like. The term preferably contemplstes thatthe molecule of interests present ina solution or composition at Teast 809% by weight: relerably, at least 85% by weights more preferably, atleast 95% by weight; and, most preferably, at Teast 99.8% by weight. Water, buflers, and other small molecules, especially molecules having molecular Weight of Jess than about one kDa, can be present. ‘The term “substantially purifled”, as used herein, refers to nucleic or amino acid sequences that ae removed rom their ‘tural environment, isolated or separated, and are a lastUS 7,393,928 B2 13 60% fee, preferably 75% free, and most preferably 90% free fom ollier componcnts with which they are naturally associ ated "A “substitution” isthe replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively. The term ‘transfection’ as used herein refers to the process ‘of introducing an expression vector into a cell. Various trans fection techniques are knows in the at, for example, microinjection, lipofection or the use of « gene pu “Transformation”, as defined herein, describes a process by which exogenous mucleie acid sequences, ext, DNA, ‘entersand changesa recipient eel, Transformation may occur Under anal or artificial conditions using various methods ‘well knovsn inte at. Transformation may rely onany known, method forthe insertion of foreign nucleic acid sequences into prokaryotic or eukaryotic host cell. The method is selected based onthe type of host ell being transformed and ‘may inelude, but isnot limited t, viral infection, electroporation, beat shock, Hpofection, and particle bombardment Sch “trnsformed?” cells include stably transformed cells in Which the inserted DNA is capable of replication either as an sulonomously replicating plasmid or as part ofthe host chro- ‘mosone, and also inchide cells which trinsieaty express the Inserted nuclei acid fr limited periods of time. As used herein, the term “vaccine” refers to a preparation of killed or modified microorganisms, living sttensted ‘organisms, or Hiving folly virulent organisms, or any other agents inchuding. but not limited to peptides, proteins, biological macromofecules, or nueleie acids, natural, symthetic, for semi-symthetic, administered 1o produce or arificially ‘increase immunity o a particular disease in order to prevent future infection with a similar entity. Vaccines can contain live, or inaetive microorganisms, or other agents, including viruses and bacteria, as wel as subunit, synthetic, semi-syn- thetie, or recombinant DNA-based nes ean be monovalent (a single strain/microorgan- unisease vaceine) consisting of one microorganism or agent (¢. poliovirus vaccine) or the antigens of one micro- ‘organism oF agent. Vaccines can also be multivalent, ©, slivalea, trivalent, etc. (a combined vaccine), consisting of ‘more than one microorganism or agent (@.2, measles- ‘mumps-rubella (MMR) vaccine) or the antigens of more than ‘one microorganism or agent. ive vaceines are prepared from living microorganisms Artenuated vaccines ae five vaceines prepared fom micro= ‘organisms which have undergone physical alteration (suchas radiation oF temperature conditioning) or serial passage in Jaboratorysnimal hosts o infected tssuefcell cultures, such treatments producing avirulent sess or strains of reduced virulence but maintaining the capability of inducing protective immunity. Examples of live atenusted vaccines include measles, mumps, rbells, and canine distemper. Inactivated ‘vaccines are vaccines in which the infectious mierobial com ponents have been desired, e.g, by chemical or physical treatment (such as formalin, beta-propiolacione or gamma radiation), without affecting the antigenicity or immunoge- icity ofthe viral coat or bacterial outer membrane proteins. Examples of iagetivated or subunit vaccines include inli- ‘za, Hepatitis A, and poliomyelitis (IPV) vaccines. ‘Subunit vaccines are composed of key macromolecules from, e, the viral, bacterial, or other agent responsible for ‘lieing an immune response. These components ean be ‘obtained in a number of ways, for example, through purfica tion from microorganisms, generation using. recombinant DNA technology ete. Subunit vaccines can contain synthetic mimics of any infective agent, Subunit vaccines ean include o 14 nacromolecules such as bacterial proein toxins (eet us, diphtheria) viral proteins (eg. from influenza viru), polysaccharides from encapsulated bacteria (e.rom fae” ‘nophilus influenzae and Streptococcus pneumonia), and Viruslike particles produced by recombinant DNA technol- ‘gy (es. hepalits B surface antigen) etc ‘Synthetic vaccines are vaccines made up of small synthetic peptides that mimic the surface antigens of pathogens and are Smmunogenie, or may be vaccines manvfactared with the aid of recombinant DNA techniques. including whole viruses ‘whose nuclei acids have been modified Semi-synhetic vaccines, or conjugate vaccines, consis of polysaccharide antigens from microorganisms attached t0 protein carrier molecules. DNA vaccines contain recombinant DNA vectors encod igen, which, upon expression ofthe encoded: in bos cells having taken up the DNA, indoce humoral and cellular immune responses against the encoded antigens ‘Vaccines have been developed fora variety of infectious ‘agens. The present invention is directed to recombinant gelatins that can be wsed in vacsine formulations regardless the ‘agent involved, and are ths not Tmited o use in the vaccines specifically described herein by way of example. Vaccines fnclude, bit are not limited t, vaceines for Yacinnia virus (small pox), polio vis (Salk and Sabin), mumps, measles, rubella, diphtheria, tetanus, Varicella-Zoster (chicken pox! shingles) pertussis (whopping cough), Bucille Calmette- Guerin (BCG, tuberculosis), aemophius influenzae menin- itis, rabies, cholera, Japanese encephalitis virus, salmonella ‘gph, shigella, hepatitis A, hepatitis B, adenovirus, yellow fever, foot-and-mouth disease herpes simplex viru, respir- {ory syncytial vias, rotavirus, Dengue, West Nile virus. Tur key herpes viris (Marek’s Disease), infitenza, and anthrax. The term vaccine as used herein includes reference to Wie cines to various infectious and autoimmune diseases and ‘angers that have been or that wll be developed, fo example, vvaceines to various infetious and autoimmune diseases and lari, and vaccines 0 breast, ung, colon, ena, bladder, and ovarian cancers. ‘A polypeptide or amino acid “variant” isan amino acid sequence that s altered by one or more amino acids from a particular amino acd sequence. A polypeptide variant may bhave conservative changes, wherein a substitated amino acid has similar structural or chemical properties othe amino acid placed, eg. replacement of leucine wth isoleucine. A vari ‘ant may’ also have nonconserative changes, in which the substituted amino acid has physical properties different from ‘those ofthe replaced amino acid, eg. replacement of a gly cine witha tryptophan. Analogous minor variations may also include amine acid deletions or insertions, or both. Prefer ably, amino acid variants retain certain structural or func- ‘ional characteristics ofa particular polypeptide. Guidance in eleriining which amino acid residues may be substituted inserted, or deleted may be found, for example, using com: puter programs well known in the ar, such as LASERGENE software (DNASTAR Ine., Madison, Wis) ‘A polynucleotide variant isa variant of a particular poly ‘nucletide sequence that preferably has atleast about 80%, nore preferably atleast about 909%, and most preferably a least about 95% polynucleotide sequence similarity 1 the particular polynucleotide sequence. It wil be appreciated by {hose skilled in the at that as result of the degeneracy of the denote code, a mulitude of variant polynucleotide soquences encoding a particular protein, some bearing minimal homol- ‘gy tothe polynucleotide sequences of any known and nat rly occurring gene, may he prodiced. Ths, the invention ‘contemplates each and every possible variation of polymucle-US 7,393,928 B2 15 ‘tide sequence that could be made by selecting combinations bused on possible codon choices, These combinations are made in abcondance withthe standard codon teiplet genetic ‘code, and all such variations are to be considered as being specially disclosed, INVENTION ‘The present invention provides recombinant gelatins and ‘methods for producing these gelatins. The recombinant gelatins ofthe present invention provide consistent and improved performance, and are able to adress various health and other ‘concerns. Using the present methods, gelatin ean be directly ‘manufactured, rather than extracted from animal sources through lengthy and harsh processes. The recombinant gels tin ofthe present iaventioa is free of pathogens, for example, pathogenic bacteria, transmissible spongitorm eneephalo thies (TSFs) ete, The present methods minimize variability and allow fora degroe of reprociibility unattainable in cure Fent extraction methods ‘Safety issues, such as concern over potential immuno onic, eg. antigenic and allerenie, responses, have arisen regarding the use of animal-derived produels. The inability to ‘completely characterize, purify, or reproduce animal-source pelatin mixtures used currently is of ongoing concern in the pharmaceutical and medical communities. Aditonal safety ‘concems exist with respect to hacterial contamination and ‘endotoxin load resulting from theextraction and purification processes ‘The recombinant gelatns of the preset invention adress these concerns as they are virally Tree of bacterial conta nation or endotoxins. Furthermore, the recombinant human tgelatinso the presen invention will offer distinct advantages ‘over animal-derived counterparts curently in use, 2 the Use ‘of gelatns derived from native human sequence ean eliminate the risk of immune response de tothe use of non-human, ‘animal-derived proteins Inadton, the present gelatin ean be produced as various ‘and distinct materials, with characteristics optimized for particular applications. ‘The resultant products are internally ‘more vonssieat and uniform than are currealy available ‘latins derived from animal sources, In one embodiment, the present invention provides 3 recombinant gelatin. The gelatin can be prodiced using various species including. but not Timited 0, - poten, equine, rodent, chicken, ovine, and piscine species, or from non-vertebrate species, The gelatin of the present invention has increased purity as compared w the gelatin products ofcurent methods of manufscture, and has 8 reduced protein load and reduced levels of endotoxins and ‘other contaminants, including nuclei acids, polysaccharides, prions ete. The present gelatin is thus saferto use than gelatin ‘manufactured by current methods, and ean be administered t0 ‘or ingested by humans and animals ata higher dosage while minimizing the risk of negative side effects ‘The gelatin ofthe present invention have increased activ ity and workability compared to commercial gelatins, a the present gelatin can be produced directly with characterises ‘optimized for specific ses, improving one’s ability to wseand ‘ormulate the gelatin. While gelatins curently extracted rom simul sources are eterogencous products witha wide rage in molecular weights throughout given batch or sample, the telains ofthe present invention include consistent, homogeneous, and repexiueble products, “The recombinant gelatins of the present iavention ean be produced using a variety of methods. In one micthod, the elatin is produced through processing of 0 o 16 recombinant collagen, (See, e., Examples 9, 10, nd 11.) ‘another method, the recombinant gelatin is produced directly trom the expression oF altered collagen constnicts, i, on- structs coalsining a polynucleotide encoding at least one collagenous domain, but not encoding naturally occurring collagen, (See, eg, Fxamples I, 4, and 6, Inanother aspect, the recombinant gelatin is derived from polypeptides which are not full-length naturally occurring collagen or procollagen, but which contain atleast one collagenous domain. (See, 2, SEQ ID NOs:15 through 25, 30, 31, and 33.) ‘Recombinant gelatns can also comprise sequences containing additional N-terminal or C-temninal propeptides. (Seo, eg, SEQ ID NOs:26 through 29.) Tnone aspect, the recombinant gelatin ofthe present iven- sion is derived from recombinant collagens or procollegens Collagen molecules generally result from trimeric assembly ‘of polypeptide chains containing (Gly-X-Y-), repeats whieh allow feethe formation of triple helical domains under oral biological conditions. See, eg., van der Rest etal, (1991), FASEB J. $:2814-2823,) At present, about twenty distinet collagen types have been identified in vertebrates, including bovine, ovine, porcine, chicken und human collagens. A atsiled description of stricture and biological fnctions of the various types of naturally occurring collagens ean be found, among other places, in Ayad ot al, The Extrcclular Matrix Foets Book, Academie Press, San Diego, Cali. Burgeson, RE, and. Nimmi ‘Molecular Steuenre and Tissue Distbution," Clin, Onhop. 282:250-272; Kelty, C. M. etal. (1993) “The Collagen Fam- lly: Sircture, Assembly And Organization In The Extracel- Jular Matrix in Connective Tissue And Its Heritable Disor- ers, Moleculae Genetis, And Medical Aspeets, Royee, P.M. and Steinmann, B., Fds. Wiley-Liss, NY, pp. 103-147; and Prockop and Kivirikko (1995) *Collagens: Molectlar biol- ‘ogy; diseases, and potentials for therapy”. Annu Rev Biochem 6403-434 Type [collagen isthe major fibrillar collagen of bone snd skin, comprising approximately 80.90% of an organism's {otal collagen. Type I collagen is the major structural maero- molecule present inthe extacelular matrix of multicellular ‘organisms and comprises approximately 20% of total protein muss. Type I collagen isa heterotrimeric molecule compeis- ‘ng two a) chains and one «2(D chain, whieh are encoded by the COLIAL and COLIA2 genes, respectively. Other collagen types are less abundant than type collagen and ‘exhibit diferent distribution patterns. For example, ype Il collagens the predominancollageninestilage and vitreous ‘humor, while type Il collagens found at high levels in blood vessels and to lesser extent in skin. ype Ill collagen is major fibriflar collagen found in skin and vascular tissues. Type IIT collagen is a homotrimesic collagen comprising three identical 1 (I1) chains encoded by the COL3AI gene. Methods for purifying various eol- Jagens from tissues ean he found, for example, in, Byers ta (1974) Biochemistry 13:5243-5248; and Miller and Rhodes (1982) Methods in Enzymology 8233-64 Posttranslaional enzymes ae important to the biosynthe= sis of procollagens and collagens. For example, prolyl 4hy- ddroxylase is a post-traslational enzyme necessary for the synthesis of procollagen or collagen by eels. This enzyme hhydroxylates prolyl residues in the Y-position of repeating Gly-X-¥ soquences fo 4-hydroxyproine. (Seo, e.g, rockon tal. (1984) N. Engl. J. Mod. 311:376-386, Unless an appropriate number of ¥position proly esidues are hydroxylated fo d-hydroxyproline by prolyl 4-hydroxylase, the newly synthesized chains canaot mainisin a stable iple-helical conforUS 7,393,928 B2 17 mation. Moreover, if no hydroxylation or uader-hydroxylation occurs, the polypeptides are not secreted properly and may be degenerated. ‘estebrate prolyl 4-hydroxylase isan aps tetramer (See, ‘eg. Berg and Prockop (1973) J. Biol Chem. 248:1175-1192;, ‘and Tuderman et al. (1975) Bur, J. Biochem. 52:9-16,) Thea. subunits contain the catalytic sites involved in the hydroxy lation of prolyl residues, but are insoluble in te absence off subunits. The subunits, protein disulfide isomerase, cata- |yzethiolisulide interchanges leading to formation of ise ulfde bonds essential. establishing a stable protein. The subunits retain 50% of protein disulfide isomerase activity ‘when part of the profyl 4-hydroxylase tetramer. (See, © Pihlajniem etal. (1987) Fmbo J, 6:643-649; Parkkonen et al. (1988) Biochem. J. 256:1005-1011; and Koivu e€ al, (1987) J. Biol. Chem. 262:6447-6449.) Active recombinant human prolyl 4-hydroxylase has been, producedin, eg, S19 insect cells andin yeast cells, by imul- taneously expressing the et and subunits (See, e.g, Voor et al. (1992) Proe. Natl. Acad. Sci, USA. 89:7467-7470; US, Put. No. §,593,859,) In addition to prolyl 4-hydroxylase ‘other collagen post-iranslational enzymes have Been identi fied and reported in the literature, including C-proteinase, Neproteinse,Iysy oxidase, Iysyl hydroxylase cle (See. e.g ‘Olsen ot al. (1991) Cell Biology of Extracellular Matrix, 2° ed, Fay editor, Plenum Press, New York) ‘The present invention specifically contemplates the use of ny compound, biological or chemical, that confers hydrox Jation,¢ proline hydroxylation and! or lysy1hydroxslatio ‘tc. as desired, to the present recombinant gelatins. This includes, for example, proyl 4-bydroxylase from any spe- ‘ies, endogenously or exogenously supplied, including vari- ‘ous isoforms of prolyl 4-hydroxylase and any variants oF fingments or subunits of prolyl hydroxylase having the desired activity, whether native, synthetic, or semi-synthetic, tnd other bydroxylases such as prolyl 3-hydroxylase, ec (Seo, e.g, US. Pat. No. 5,928,922, incorporated by reference herein in ts entirety) Inomcembodiment the proly hydroxy Jase activity iseoniered by aprolythydroxylase derived rom the same species as the polyicleotide encoding recombinant eatin or encoding a polypeptide from whieh recombinant telatin can be derive. In further embodiment dhe prolyl ‘{hydeoxylaso is human and the encoding polynucleotide is ‘rived from human sequence. ‘The present invention provides methods for manipulating the thermoplastcity of gelatin in onder to produce a material with the desired physical characteristics In ome method, the ‘encoding polynucleotides are expressed ina host system hav= ing endogenous prolyl hydroxylase of allemate hydroxy Jases, such as contain mammalian or insect eels or transgenic ‘animals, or plants or plant cells, Ia such a system, the present invention provides methods for producing a mixture of fecombinant gelatins having a range of percentages of hydroxylation, ic, non-hydroxylated, partially hydroxy Jated, and fully hydroxylated porions. For example, in one method of producing recombinant gelatins with varying per ‘centages of hydroxylation, the hydroxylation is conferred by ‘endogenous prolyl hydroxylase in, eg. transgenic ania, andthe distribution of percentage hydroxylation ranges fom non-hydroxylated to flly-hydroxylated, and the melting temperatures of the material produced range from 28° C. 0 36°C. with a median T, value of around 30° C.t032°C. ‘desired, different fractions of the material can be isolated slong a temperature gradient, as might be nocessary if downstream uses require selecting, for example, the more fally o 18 hydroxylated materials, such a8 those sufficiently hydroxy Jated 0 retain triple helical structure at eg, body temper- ture 7" C). ‘Tn another embodiment, recombinant gelatins are produced! in a system, eg. @ transgenic animal, in whieh hydroxylation. is supplemented with exogenous pralyl hydroxylase. In one aspect, such a method of producing recombinant gelatns provides recombinant gelatin ranging from non-hydeoxylatedto fully-bydroxylated. The fraction of recombinant gelatine more fully hydroxylated will be sub- ‘Stanvially larger in recombinant material produced in the pees- fence of exogenous prolyl hydroxylase than in recombinant ‘material produced only inthe resenee of endogenous prolyl hydroxylase. Therefore, the melting. temperatures of the ‘material produced ean range from, for example, 28° C.t0 40° Caving a median T,, value of around 34°C, 10 36°C. Such «gelatin mixture could be appropriate fr use ina variety of applications, such as gel capsule manufacture, without requiring any fractionation or separation of differently hydroxylated portions "The above methods prove for production of recombinant materials with a ringe of melting temperatures, that can be casly divided, for example, using a temperature gradient to Separate materials solid ata particular temperature, ¢., 36° from those liquid at a particular temperature. Further mon, the present invention provide for cost-effective meth- ‘ods of producing a material which, without separation, is suitable for use in bulk applications. For example, the man Tactre of gel eapsules could involve the use of recombinant gelatin prcioced by the above methods, wherein the recombinant material, having a range of melting temperatures, had ‘adesirablemeitingemperatirof round 33°C.,sueh gelatin relting at body temperatures, and thus being stitable for swallowing and digestion. Inthe present methods, the recombinant gelatin can be produced dirty in the desired system, eg. transgenic aaimal, or ean be derived, for example, through hydrolysis, eg. acid, thermal, or enzymatic, from combinant collagens produced inthe desired syste, In ene embodiment, the present invention provides a method of producing recombinant gelatin comprising pro- ‘dacing recombinant collagen and deriving recombinant gelatin from the recombinant collagen. In one aspect, the method comprises the expression of at least one polynucleotide sequence encoding a collagen or procollagen, or fragment or Variant there, aad at lest one polynucleotide encoding « collagen post-iranslational enzyme ora subunit thereof. (See, eg, US. Pat. No. 5,593,859, incorporated by reference Doerein in its entirety) The present recombinant gelatins ean be derived from recombinant collagens using: procedures Known in the art. (See, eg., Veis (1965) Int Rev Connect issueRes,3:113-200,) For example, acommoa feature ofall collagen-(o-gelatin extraction processes is the loss of the secondary structure ofthe collagen proein, and inthe major- ity of instances, an alteration in collagen structure. The eo! Jagens used in producing the glatns of the present invention canbe processed using diferent procedures depending onthe type of gelatin desired. Gelatin of the present invention can be derived from recombinantly produced collagen, or procollagens or other collagenous polypeptides, or fom cell cultures, eg, verte- bate cell cultures, by a variety of methods known inthe ar. For example, gelatin may be derived dirty from the col ‘mass or the culture medium by taking advantage of gelatn’s solubility at elevated temperatures and is stability under con- itions oFlow orhighpH, low orhigh alt concenirations, and high temperatures. Methods, processes, and techniques of producing gelatin compositions from collagen include diges-US 7,393,928 B2 19 tion with proteolytic enzymes elevated temperatures denaturing the wiple helical structure of the collagen utilizing ‘detergents, heat, or various denaturing agents well known i the ar, ec. In addition, various steps involved inthe extrac- tionof gelatin from animal or slaughterhouse sources, include Jing treatment with lime of acids, heat extraction ia aqueous solution, ion exchange cimmatograph. eross-Row iron, and various methods of drying can be used 10 derive the _elatn ofthe present invention from recombinaat collagen In one aspect, the gelatin ofthe present invention is com= prised of denatured tiple helices, and comprises a least one ‘collagen subunit, colligen chain. or fragment thereof. The ‘Gly-X-Y units within particular collagen chain, subunit, oF Jagment thereof may be the same or different. Preferably. X and ¥ are either proline or hydroxyproline, and glycine ‘appears in about every third residue position ofthe component chain, Theamino acids oF X and are proline oF hydox- -yproline, and each Gly-X-Y units the same o different. In nother embodiment, the recombinant gelatin of the present Jnvention comprises an amino acid sequence of (GIy-X-Y x wherein X and’Y are any amine acid. In one embodiment, the present gelatin is derived from 3 recombinant collagen of one type tht is substantially free from collagen of any other collagen type. In one aspect, the recombinant collazens typeI collagen. Inanother aspect, the recombinant collagen is type Il collagen, In another mbox ‘ment of the present invention, the recombinant collagen is human recombinant collagen. Puntner embodiments of the inventioa, in which the revombinant collagen is of ay one collagen iype, such at any one of collagen types through XX, inclusively, or any other collagen, natura, synthetic, oF semi-synthetic, are specifically contemplated, Embodiments jn which the recombinant gelatin is derived from specified mixtures of anyone ormoreof any ofcollagen ypes I through XX, inelnsvely, or any other collagen, natural, synthetic, oF semi-synthetc, ar specifically contemplated ‘The present methods of producing recombinant gelatin have a number of advantages over traditional methods of tzlatin extrvction. Most importanly, the present methods provide a reliable non-tssue source of gelatin contining native collagen sequence. In addition, curreat methods of cexirction donot allow for any tural source of human telain, such as might be advantageous for use in various medical applications. The present invention specifically provides recombinant gelatins derived from human sequences ‘compositions comprising recombinant human gelatns, and ‘thods of producing these gelatins, Therecombinant human gelatin jsnon-immonogenie as applied in pharmaceutical and medical processes, and various uses thereof are also contemplated. In another aspect, the preset invention provides for the luctioa ofthe resent gelatin fom engineered consieuts ‘capable expressing gelatin in various forms. This invention specifially contemplates methods of producing gelatin sing recombinant prolyl hydroxylase and various synthetic con- ‘ruc, including non-native collagen constructs, Further, the present invention provides recombinant gelatin that can be ‘esined (0 possess the specific characteristics needed fora Particular application. Methods for producing these gelatins are also contemplated. Using the eurent methods, one could produce 2 golatin with the desired gel strength, viscosity. melting characteristics, isoelectric profile, pH, degree of hydroxylation, amino-aeid composition, odor, color, ete. In ‘one method according to the present invention, non-hydo- lyzed gelatin is produced, and can be subsequently hydro- Jae fully or partially, if desire. 0 o 20 Properties of Gel The varius physical properties of gelatin define its usefulness in particular applications. Gelatin provides unique performance based on, for example, its amphoteric nature, its ability to form themo- reversible gels, its protective colloidal and surface active properties, and its contribution to viscosity and stability. Ina number of applications, gelatin is used, for ‘example, san emulsifier thickener or stabilizer, asan agent {or film o coating formation a a binding agent as an athe- sive or glue; oras a locculating agent ‘Raw materials, (ypes of pre-teatmeat, and extraction peo= cesses all effect the composition of gelatin polypeptides ‘oblained during conventional mannactre. Currently 2 ‘le animal prods re thus heterogeneous protein mixtures ‘of polypeptide chains. Gelatin molecules can be fuel Iarye, olecular weight within particular sample ranging from a few to several hundred kDa, The molecular weight distribution of gelatin in a particular lt can be ertical, as ‘weight distribution can influence, for example, the viscosity andor gl strength ofa gelatin semspe, ‘In general the viscosity of a gelatin solution increases with increasing concentration and with decreasing temperature, A higher viscosity solution would be prefered, for example, for gelatin used asa stabilize or thickener. In some applications, Jiquid geatins are preferred, soch asin various emulsifying ‘uid, ete. Viscosity of a gelatin solution increases. with increasing molecular weight of the gelatin components. A hiph-viscosity gelatin solution could consist, therefore, of high concentration of low molecular weight gelatins, or of a lower concentration of high molecular weight gelatins. Vi cosity also affects gel properties including seting and me ing point. High-viscosity gelatin solutions provide gels with higher melting nd setting rates than do lower viscosity gelatin solutions ‘The hermoreversbility and thermoplasticity of gelatin are properties exploited ina numberof applications, for example, inthe manfactore of gel eapsles and tablets. Gelatin can be beated, molded or shaped 2s appropriate, and cooled to form 4 capsule or tablet coating that bas unique properties at homeostatic temperatures. The gelatin will begin to melt at south temperature, easing swallossing, and bocome liquid at body temperatures. Gelatins of various gel strengths are suitable for we in erent applications. The fizmness or strength of the set gel is typically measured by calculating the Bloom value, whieh «an be determined using intemational standards and method: ology. Briefly the Bloom strength is a measurement of the strength of gel formed by’a 6.67% solution of gelatin in a ‘constant temperature bath over 18 hours. standard Texture ‘Analyzer is used to measure the weight in grams required to depress a standard AOAC (Association of Official Agricul tural Chemists) plunger 4 millimeters into the gel. I the ‘Weight in grams required for depression of tse plunger is 200 rams, the panicular gelatin has a Bloom value of 200. (Seo, g.. United States Pharmacopocia and Oficial Methods of Analysis of AOAC International, 1 edition, Volume Il) ‘Commercial latins can ths be graded and sold on Bloom strength, Different rangesof Bloom values are appeopeite for iferent uses of gelatin; for example, gelatine for use various industrial applications, eg, conerete stabilization, sand easing, molds, glues, coatings, ee, will be selected froma wide range of varying loom strengths, depending on the performance characterises desired. Gelatns with vary~ ‘ng Bloom strengths are also desired in the manufacture of various pharmaceutieal products. Por example, sot el cap- sles are typically manefactured using ossein or skin gelatin ‘with @ Bloom value of sbout 150 t0 175 aadior porcine:US 7,393,928 B2 2 derived gelatin with Bloom valve of about 190 0 210, of Blends derof, while hard gel capsules jet use a glain ‘ith Bloom value faut 220 0 260, In foe applications, latin ved, for example, thickener in marshmallows or ‘ther confectionary proses might havea Bloom seat of sownd 250, Varios applications, ischading certin ess {ying vids n photographic applications and various inds- al eotigssavlve the use of nongeling plains The present invetion provides Tor the peoction of recombinant glans with diffrent Blooms statis In oe ‘spect the present invention provides, for example, for the ‘anual of gelatin with Blom strengths of around 50 100, 150,20, 250, and 300 none embrstimeat,the present invention provides forthe prodocten ofa recombinant gelae inhaving’a Bloom strength of aronnd 40 Such glatinean bused, for example, in Ue mannfacture of gel capsules, and ‘could alow forthe manufature of a lighter and thinner ‘capsule, as less material would nee to be wid o provide el of sufiient stent. Recombinant geltins wih Bloom Strengths of wader 100, and fom Oto 100, inclusively, re also ‘contemplated “The present invention provides methods for designing recombinant gelatin with he pysical properties desi oe particular applications. In one embodiment, the present Invention provides recombinant gelatins comprising unifom > molecules of a specified molecular weight or range of molecular weights, and methods for producing these recombinant gelatins. Such homogeneous and uniform materials ‘advantagsous in that they provide a reliable source of produet with predictable performanee, minimizing vaiabil Sty in product performance and in mansfacturing parameters. Currently, gelatin from different lots must sometimes be blended in order to prodice a mixture with the desired physi ‘al characteristic, such asthe viscosity of gel steengt, ee, provided by a particular molecular weight or molecular ‘weight range. Tn applications in whicha specifi molecular weight range ‘of recombinant gelatin won be preferred to-a recombinant gelatin withaspecific molecular weight. the present invention provides such materials, Using the recombinant gelatins of the present invention, amanufactrer could for example, mix recombinant gelatine from ols with specified molecular ‘weights, in certain percentages, inonder to achieve a mixture with the desired molecular weight range. Additionally, the present recombinant gelatins are inherently more uniform ‘and of greater consistency than currently available commer. ial prodlets. none method of the present invention, recombinant collagen is processed, such is by acid or heat hydeoly- sis, to price recombinant gelatin of @ molecular weight range narrower than that of currently available gelatin products, Using suitable and controllable hydrolysis conditions, the present methods produced recombinant human gelatine with molecular weight distributions similar to those of com- ‘mercially available gelatin as well as recombinant gelatins with ranges narrower than those of the molecular weight rages of cen erase prot (oe romp 9nd The present invention provides recombinant gelains of Uniform molecular weight or specified ranges of molecular weights, removing variability and unpredictability, and allowing for fine-tuning of processes and predictable belay jor. The present methods allow for the production of recom binant geatins of any desired molecular weight or range of molecular weights. For example, in one embodiment, the recombinant gelatin has a molecular weight greater than 300 Ds, In another embodiment, the recombinant gelatin has & molecular weight range of from about 150 to 250 kDa, or of 2 {rom about 250 10 380 kDa. Other molecular weight ranges are specifically contemplated, including, but not limited to, the following molecular weight ranges: about O 1 50 kDa, bout $0 © 100 kDa, about 10 t0 150 kDa, about 150 9 200 kDa, about 200 0 250 kDa, about 250 1 300 kDa, and about 300 10 350 kDa, ‘In another aspect, recombinant gelatin with a molecular ‘weight similar to that of some commercially available gelatins, of from about 10 to 70 kDa, could he produced. In preferred embodiments, the present invention provides recombinant gelatin narrower molecular Weight ranges, not curently available in commercial produets, such as Irom about 10 f0 30 kDa, shout 30 19 50 KDs, and about 50 10 70 kDa. In a particular embodiment, a recombinant gelatin With ‘8 chain length conferring specific properties appropriate to the intended application s provided. In various embodiments fof the present invention, recombinant gelains with uniform ‘molecular weights of approximately 1 kDa, § kDa, 8 kDa, 9 KDa,14kDa, 16kDa,22kDa, 23 kDa, 44 kDa, and 6SkDaare contemplated. (See, eg, Table 2.) TInpartcular, in one method ofthe present invention, gel tin #8 produced Irom shortened collagen sequences, Tor example, the sequences identified in Table 2. These sequences represent specific collagenous domains and encode short forms of gelatin The present gelatins are capable of retaining valuable physical characteristics of gelatin, for example, ilm-fomning abilities, while possessing average molecular weighs lower ‘orhigher than those of conventionally derived animal gelatin. ‘Various modifications of collagen sequences, ineiing, for example, denaturing ofthe collagen, collagen chain, subunit or fragments thereof, or varying degrees of hydroxylation fain be made that will podice gelatin with specific physical properties, ie, 2 higher or ower melting point than conven- ‘ional gelatin, different amino acid compositions specific molecular weights or ranges of molecular weights, ete, and stich variations are specifically contemplated her ‘The molecular weight ofa typical fibil-forming collagen molecule, such as type I collagen, is 300 kDa. In some ap cations, suchas those in whieh high molecular weight ge tins are used, itmight he desirable to produce a gelatin with a ateater molecular weight than that of currently available fextracied gelatin. Therefore, in one embodiment of the present invention, gelatin ean be produced containing mol- ‘ecules larger than the collagen from which commercial gelatin is currently extracted. The resultant higher molecular ‘weight gelatin produet can be used directly in various appl «ations in which its physical properties would be desirable, or can be divided and subsequent treated to produce molecules ofa smaller sizes. In one embodiment, gelatin can be produced using col- Jagens larger than those available in conventional animal sources, For example, the preseat methods of production could be adapted to produce the acid-soluble cutiele collagens derived from the body walls of vestimentiferan tbe worm Rifia pachypila (molecular weight-2600 kDa) and anncid Afvinelfa pompejana (molecular weight~1700 kDa). These collagens could be adapted to the present methods of production to produce larger molecules than those fr ‘whieh currently available gelatin i extracted, andthe resul- ant product could be tested to produce peatins as desired It is specifically contemplated that gelatins of various ‘molecular weights ean be produce by a variety of methods according to the prosont invention. For example, characters- ties of the present recombinant gelatins, eg, percentage hydroxylation, degrees of cross-linking, ct..can he varied to produce recominiigelatins with the desired molecularUS 7,393,928 B2 23 weights. In ove aspect, For example, the present inventio provides a method for producing large molecular weight ecombiniant gelatns by using eoss-inking agents know in the art to cross-link gelatin polypeptides. (See discussion, ingin) In another aspect of the present invention, polypeptides from which gelatins could be derived are expressed from ‘engincered constricts containing multiple copies of all of fragments of native collagen sequence. For example, in one ‘embodiment, he present invention provides an altered col Tagen construct comprising multiple copies of the collag- ‘enous domain of type collagen. In another embodiment, the ‘eonstruet comprises multiple copies of the collagenous ‘domain of type TIT eollggen. Ina futher embodiment, the ‘construct comprises copies of type Tand type Il collagenous ‘domains. The present invention provides forthe use of single ‘or muiple copies ofall or portions of sequences encoding any collagen, including collages type I through XX, inela- sive. Its specifically contemplated that the present methods allow forthe production of gelains derived from more tha, ‘one ype of collagen. In one emibodiment, recombinant gla tins derived from more than one type of collagen are o- ‘expressed inan expression system ¢g ,a bos cel, transgenic ‘animal, ef, stich that a mixture of glatins is produced, In another embodiment, the present invention provides 3 ‘method for producing gelatin without derivation from a eollagen or procollagen tiple helical stage. In one aspect, this involves produetion of recombinant gelatin by expression of ‘varius constrets ina high-temperature expression system, such as one relying on thermophilic organisms, that does not low the formation of triple helical strictares, but permits the ‘activity of proly hydroxylase. The preseat gelatin could also be derived from collagen constmicts containing mutations, ‘additions, or deletions that prevent tiple helical formation. I fanother aspect this involves production of gelatin from short- ‘ene! construcis that do not allow for formation of tiple helices at regular temperature, i.e, 37° C. Altematively, gelatin can be produced inthe presence of inhibitors of tiple helix formation, for example, polyanioas, that are co-expressed with the biosynthetic collagen consteucts. Addition- ally the biosysthetic gelatin ofthe present invention could be ‘derived om recombinantly proiced collagen chins that do not fora triple helices. TInanother embodiment, the iavention provides a method of deriving gelatin from non-hydroxylated collagen or collagen, Jn which there is partial rather than fll hydroxylation of proline residues. In one aspect, this method comprises deriv- Ing gelatin from collagen expressed inthe absence of prolyl hydroxylase, for example, in an inseet expression system ‘without prolyl hydroxylase. (See, eg, Myllyarju et al (1997) J. Biol. Chem. 272, 21824-21880.) In one method according to the present invention, gelatin is derived from the parially hydroxylated or non-hydroxylated collagen. Fiydroxylation ean be conferred, for example, by in vitro ‘administration of hydroxylase. none method, alow degree ‘fsubstitution of hydroxyproline for prone can be foreed by providing hydroxyproline to, eg., bacterial or yeast host cals The present invention comprises fully-bydroxylated, partially-hydroxylated, and non-hydroxylated recombinant elatins. In anther embodiment, the method of the present ‘vention comprises podacing a gelatin or gelatin precursor having specific degree of hydroxylation In further aspect, the invention relates to a method of producing gelatin having from 20 1 80 percent hydroxylation, preferably, from about 30 to 60 percent hydroxylation, and, most preferably, about 40 percent hydroxylation. (See Examples 4 and 5.) The par- 0 o 2 ‘illy-hydoxylated recombinant gelatias of the prese invention can be obtained though mixing specified pervent- ages of recombinant gelatins with different degrees of hydroxylation, or can be obtained directly. ‘See Examples 4 and 5.) Further, the invention provides methods for achieving partial hydroxylation of recombinant gelatins by administer ing prolyl hydroxylase to non-hydroxylated_ recombinant agelatins in vitro, and controlling the length of the reaction. There are limits o the extent to which the thermal charac teristics of curently available animal-source gelatins can be altered. The present invention specifically provides for meth- fds of producing recombinant gelatin, wherein the recom! ‘nant gelatin has the specific thermal charseteristies desired {orapanicular application, Using the methods ofthe present invention, for example, the melting point andor gel strength ofthe recombinant gelatin can be manipulated ina variety of ways. The emperatre stability anor ge strength of recom binant gelatin can be measured by a variety of techniques well-known inthe ar ‘Generally, the melting point of gelatin increases as the degree of hydroxylation increases. Using the methods ofthe present invention, it i possible o produce high molecular ‘Weight gelatine that, de to manipblation of hydroxylation andor cross-linking, et., have a lower gel strength andor lower melting point than those of curently available animal- source gelatins. Therefore, the present invention provides 3 recombinant gelatin with properties unsttsinable in various ‘commercial produets, suitable fr usein applications where a higher molecular weight gelatin is desired, in order‘o provide ‘increased film strength te, but a non-gelling or low strength gel products desired, Inone embodiment, the present invention provides recombinant gelatin that has lower temperature stability de to incomplete hydoyation of proline resis. Such a recombinant gelatin could be usefUl ina variety of applications, In gelatin produced by current extraction meth- fod, only fish gelatin provides a high average molecular ‘weight film-forming protein that is non-geling. ‘The non- gelling and cold watersolubility characteristics offered by ‘bon-geling fish gelatin can he matched by curently available hydrolyzed bovine and porcine glatins, but with correspond ing loss of film strength and Hexibilty, asthe hydrolyzed aelatns are of ower average molecular weight. Therefore, in ‘one embodiment, the present invention provides a partially hydroxylated ecombinant gelatin with lower gel strength and higher molecular weight than that provided by currently available animal-source materials ‘A igher molecular weight, lower gel strength recombinant gelatin could also be useful in various pharmaceutieal appi- ations, in whieh stability is desired, but oa- or low-gelling properties are desired in order to maintain the malleability fad intgrityof the pharmaceutical predict. Suc a rom ‘nant gelatin could beused,frexample, asa plasma expander, ‘sits molecular weight could provide stability. increasing the residence time in circulation, and the altered soting point ‘would prevent the material from gelling at room temperature allowing the expander to be administered without warming. In one embodiment, the present invention provides a par- sially-hydoxylated recombinant gelatin suitable for use in phamaceutical applications, for example, as a plas expander, Tn. another aspect, partilly-hydroxylated recombinant gelatin is obtained through expression of recombinant gelatin, or expression of polypeptides from which the present recombinant golatin can Be derived, inthe absence of pray hydroxylase, for example, in an insect expression system without proiy! hydroxylase. (See, eg, Myliyhariu etal (1997) J Biol. Chem. 272, 21824-21830,) Hydroxylation eanUS 7,393,928 B2 25 ‘occur at the time of production or ean be subsequeatly imposed through, ein vitro biological or chemical modic fication. none method of the present invention, recombinant _gzelatinsarederived fom prtally-hydroxylatedo rom fly hydroxylated collagen ‘Gelatins derived from natural sources by curently av able methods are greatly strengthened by the existence of ‘covalent cross-links between Iysine resides ofthe constitt- ‘ent collagen molecules. Cros-inking occurs naturally in the ‘extracellular space following collagen secretion and fibril formation, as prior to seeretion, certain Iysine resides are hydroxslaed by theenyme ss hydroxylase, The exteacel- Jular enzyme Iysyl oxidase subsequently deamidates cert Iysine and hydroxylysine resides inthe collagen molecules, ‘Yielding highly reactive aldehyde groups that reset spontane= ‘uy to form covalent bonds. The resulting crosslinked col. Jagens yield gelatns of increased gel srength and increased Viscosity. Specifically, a higher degree of crosslinking results in gelatins with higher melting temperatures and reater uel strength, Tone aspect, the preset invention provides recombinant clans that are eos-inked, resulting i higher molecule ‘weight gelatins. (See Example 7.) Cross-linking can be imposed by different methods, sich a by biological or ‘chemical mosifiation. For example, in one embodiment, Fecombinant gelatin or a polypeptide fo which gelatin a be derived is expressed inthe presence of lysyl hydroxylase ‘and sy] oxidase. In another embestiment, the polypeptides modified by cross-linking afler expresion. In a farher ‘aspect the present invention provides for imposition of erosslinking by chemical means, such as by reactive chemical ‘eroslinkers, for example I-ethyl-3-dimethylaminopropy) ‘earhodiimide hydrochloride (EDC), (See Example 7) Othe chemical crosslinking agents, such as bissulfostei ‘yl suberate (8.3. -dithibis(sulfosuccinizidy) prop ‘nate (OTSSP), and Tris-sulfosvecinimidy! aminoteaeette (Galfo-TSAT) may also be used, as can various agen known inthe an, Additionally, the present invention provides meth- ‘ods of producing recombinant glatns with varying degrees ‘ferosslnking, useful forobiaining recombinant gelatin of ‘desired melting points, gel strength, and viscosity The present invention provides methods fo manipulate the molecular weit, he evel of hydroxylation, and the dewree ‘of erosslinking ofthe recombinant geatns tallow for ere= jon of recombinant gelatns of different and speci Bloom, strengths, as well as recombinant gelatns of diferent and spovitic levels of viseosy Proline hydroxylation plays central role in natural otlagen formation, Hydroxylaton of specific Iysyl residues in the sequence X-Lys-ily also performs an important funetion in collagen synthesis and fibril formation. The hyaoxy groups ‘on modified lysine residues fanetion as both attachment sites, for carbohydrates and as essential sites forthe formation of stable intermolecular cross-links. These -mosifcatons require the expression of specific enzymes, iysyThydroxslase and ysyl oxidase. “Therefore, in one aspect ofthe invention, the co-expression ‘of these enzymes with the polypeptides of the present invention is contemplated. The kene encoding Iysyl hydroxslase (Hautala et al. (1992) Genomes 13:62-69) is expressed in a hostcel, whic isthen further mxiied by the introduction of a sequence encoding a gelatin or polypeptide from which Belatin canbe derived, as described in the present invention. ‘The recombinant gelatin ofthe present invention ean there= fore be post-ranslationaly moxie by the aetvity of endos- ‘enously expressed ysyl hydroxylase and Iysyl oxidase. The recombinant pelts ofthe present invention ean also be o 26 voditied by the expression of exogenous Iysyl hydroxylase ‘and Iysyl oxidase. In one embodiment, recombinant geatins produced are non-hydroxylate, and subsequently altered by Imposing the desired degree of hydroxylation of lysine re dues by the enzymatie activity of Iysyl hydroxylase. The ability toalterthe degree of iysy hydroxylationisdesirablein producing gelatin, and polypeptides from which gelatin can be derived, with various degrees of cross-linking that lead to the desired gel strengths and viscostis. In further embodiments, a polypeptide containing hnydroxylysine residues ean also be expressedia, for example, a yeast cel, in whieh hydroxyproline is produced by the activity of prolyl hydroxylase. (See Examples 1 and 4.) ln some embodiments, the modified recombinant gelatin or polypeptice from which gelatin can he derived can be foemn- Jated anid administered to an animal or human, Unis serving as substrate forthe activities of endogenous enzymes, such as Iysyl oxidase, thus allowing the collagenous polypeptide to be incorporated into tissues ina stabilized eros-linked form. Therefore, oneaspect ofthe present invention provides forthe production of recombinant gelstins of desirable gel strengths tnd viscosity for commercial use, without the nee fr Iysy1 hydroxylase or lysyl oxidase activities. ‘The invention also provides forthe production of gelatin, having « particular gelling point. In one embodiment, the present methods provide forthe production of gelatin having 4 seting or gelling point of from 1S to 35°C. In farther ‘embodiments, the recombinant gelatin has a setting point of rom 15 to 25° C, from 25 t0 35°C., and from 20 1 30°C. a various aspects, the present invention provides recom: binant gelatin that is now-bydrolyze, fully hydrolyzed, oF hydrolyzed to varying deurees, such as gelatins that are a sixture of hydrolyzed and noa-hydrolyacd products. Addi- ‘sionally, the prevent invention provides methods of producing recombinant gelatins with varying degrees of hydrolysis. (See Fxamples 9 and 10.) Gelatin hydrosylates are typically cold water-soluble and are used in a varity of applications particularly in the pharmaceutical and food industries, in ‘whicha gelatin with non-glling properties is desirable, Gela- tin hydrolysates are used in the pharmaceutical industy in {ilm-forming agents, microencapsulation processes, arthritis ‘and joint relief formula, tabletting, and various nutritional ormulas. Inthe cosmetics industy, gelatin hydrolysates are used in shampoos and consitioner, lotions ad other foenn- Jations, including lipsticks, and in fingernail formuls, ee Gelatin hydrolysates appear as nutritional supplements in protein and energy drinks and foods; are used as fining agents Jn wine, beer, and juice clarification; and are used in the rmicroencapsulation of additives such as food Havorings and colors. Gelatin hydrosyltes are used in industrial appica- tions for their flm-forming characteristics, seh as in eo ‘ngs of eloments in semiconductor manuiacture, ete. In one embodiment of the present invention, goatin is produced from collagen sequences in Which particular native {domains have been deleted or have been added in ore t0 alter the behavior of the expressed product. The invention further contemplates methods of producing recombinant aelatin wherein the gelatin is produced direetly from an altered collagen construct, without production of an inact ‘eile helical collagen. In particular, the present invention ‘contemplates methods of prodicing recombinant gelatin ‘comprising the expression of various engineered eonstuets that do not encode standard triple helical collagen. For example, specific deletions can eliminate collagenase-e- Sponsive regions, and various regions eliciting immunogenic, eg, antigenic and allerenie, responsesUS 7,393,928 B2 27 Specie domains of various collagens have been associ ted With specific aetvities. See, eg, Shahan etal (1999) ‘Con, Tiss Res, 40:221-232; Raff et al. (2000) Human Genet 106:19-28, both of which references are incomporatod by rof= ‘erence herein in their entireties.) In particular, the present invention specifically provides for methods of producing recombinant’ gelatins derived from collagen constructs (red to climinate orto reduce or increase specific regions a collagen gene associated with a specific activity. Speciti- cally, such regions could be deleted in allo in part pro- ‘duce a gelatin lacking oF with reduced specific aetvity, oF ‘additional copies of the specific region could be added 10 produce a gelatin with enhanced activity: For example, Sequences in types [and IT collagen recognized by the «2B ‘negrin receptor on the platelet cell surface have been iden- fied. (Knight etal. (1998) J. Bil. Chem, 273:38287.33294; and Morton et al. (1997) J. Biol. Chem, 272:1 1044-11048, ‘which rlerences are incorporated by reference herein in theit entirety) In one aspect of the present investion, it is desirable to create & homogeneots gelatin composed of fragments synthesized from collagen constructs lacking platelet setivation repions-Such gelatin could be included, for example, in prod- Uels associated with anastomosis and vascular grain, ete Including coatings for tent and graft devices. Sch products ‘can be associated with deleterious side effects, for example, thrombosis, that ean develop in association with the use of sueh products as a result of the pltelet-apgreyating regions present in the collagenous product none aspect, the present vention provides fora method of producing a recombinant latin which can provide support fr cll stfachment when, Used ina stent oe similar device, but which does not include pltelet-reactive regions, thus minimizing the risk of platelet ‘aggrezation. (See Example 2.) Therefore, the present inven tion provides in one embodiment for a stent coating comprise ing recombinant gelatin. In a preferred embodiment, the recombinant gelatin is recombinant human gelatin, In some instances, such as various wound care applications it could be desirable to. provide recombinant gelatin comprising teyplophan, or hisD, which allows cells © utilize histino in place of histidine. See, eg, Hartman, S.C. and R.C. Mul- Tigan (1988) Proc. Natl. Acad, Sei.85:8047-51_)Recently, the use of visible markers has gained popularity with such mark- ‘ers as anthocyanins, alvcuronidase and its substrate GUS, ‘and luciferase and its substrate luciferin, now widely used a0 ‘only to identify transformants, butals to quantify theamount ‘of transient or stable protein expression atibutable (a spe~ tie vector system. (See, e2., Rhodes, C. etal. (1998) Methods Mol. Biol. $5:121-131.) ‘As noted above, the expression vectors for use in the present methods of production can typically comprise a marker gene that confers a sclectable phenotype on cells Usually, the selectable marker pene will encode antibiotic resistance, with suitable genes including at least one set of tenes coding for resistance tothe antibiotic spectinomyein, the streptomycin phophotransferase (SPT) gene coding fo streptomycin resistance, the neomycin phophotransferase (NPTH) gene encoding kanamycin or geneticin resistance the hygromycin resistance gene, genes coding for resistance to herbicides which act to inhibit the action of acetolatate syuthase (ALS), in particular, the sulfonylureatype herbi cides (eg. the $4 and/or Hra mutations) genes coding for resistance to herbicides which act to inhibit action of plutamine synthase, such as phophinothricin orbasta (edhe bar gene), or other similar genes known in the art. The bar rene encodes resistance io the herbicide basta, the nll gene ‘encodes resistance to the antibioties kanamyein and genet ‘in, and the ALS gene encodes resistance to the herbicide hlorsulfuron Other methods for determining which host cells, subse “quent to transformation, contin the polynucleotides ofinter- ‘est include a variety of procedures know to those of skill in the an, These procedures include, but are not limited 0, nucleic acid hybridizations, including DNA-DNA or DNA” NA hybridizations, and various protein bioassay o immu- roassay techniques including membrane, solution, or chip- ‘hased technologies for the detetion and/or quantification of polynucleotides or polypeptides Inaddition, host cll strain may be chosen which modu Jates the expression of he inserted sequences, or modifies and processes the gene product in the specific fashion desined. Such modifications (eg, glycosylation) and processing (ez. ‘cleavage of protein products may be important forthe func tionof the protein. Different host cells have characteristic and specific mechanisms forthe post-irnsational processing and ‘modification of proteins. Appropriate ell lines or host sys- 0 o 34 tems can be ehosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host eels that possess the cellular machinery for Proper processing othe primary transcrip, including various ‘mslifcaions seh as protein folding, disulfide boa forma- ‘ion, glycosylation, and phosphorylation ofthe gene product ‘may be used, Such mammalian hos cells include, bt are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, WIBR, ce ‘Specific initiation signals may also be used to achieve more efficient translation of the polynucleotides of the present jnvention. Such signals include the ATG initiation eadon and adjacent sequences. In eases where sequences encoding the present polypeptides, along with any initiation or upstream Sequences retired for translation, et, are inserted into the appropriate expression vector, no additional tanseriptional ‘or transational control signals may be needed However, in cases where only coding sequences, or portions thereof, are inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation eodon shouldbe inthe comect reading frame 10 censure translation of the entire insert. Exogenots tras tional elements and initiation codons may be of various o gins, both natural and synthetic. (See, eg. Bittner et al. (1987) Meth in Enzymol. 153:516-548.) ‘A host call of the present invention ean be infect, tran ‘ected, oF ransformed withat leat one polymueleotideencod- ing a post-transational enzyme, in addition to atleast one polynucleotide encoding a gelatin ofthe present invention or ' polypeptide from which the gelatin can be derived. Such polynuileotides include those encoding collagen post-ans- Jational enzymes, such prolyl hydroxylase, collagen gly~ cosy! transferase, C-proteinase, N-proteinase,Iysyl oxidase, orlysyl hydroxylase, and can be inserted into cells that do not naturally produce postaranslational enzymes, for example, into yous cells, of cells tht may not naturally produce sf cient amounts of postranslational enzymes, for example, various insect and mammalian cells, such that exogenous enzyme may be required to produce certain pos-translational effects. none embodiment ofthe presen invention the pos ‘translational enzyme is pry] 4-hydroxylase, and the poly- neleotide encodes the cor the subunit of prolyl hydroxy Jase, Ina prefered embodiment, polynucleotides encoding the «subunit and the f subunit Of prolyl 4-hydroxylase are inserted into cell to produce a biologically active prolyl ‘hydroxylase enzyme, co-expressed witha polynucleotide encoxling gelatin ora polypeptide from which gelatin can be Serived. ‘Te polynucleotides encoding post-translationsl enzymes may be derived from any souree, witether natural, synthetic, correcombinant. Ina prefered embodiment, the postransla. tional enzyme is derived ffom the same species as is the ‘recombinant gelatin o be produced. In one embextiment recombinant gelatin to be produced is human recombinant gelatin, and the post translational enzyme is human pray] “hydroxylase, The expressed gelatin or gelatin precursors ofthe present invention are preferably secreted into culture media and ean be purified to homogeneity by methods known in the at, for example, by chromatography. Inone embodiment, therecombinant gelatin or gelatin precursors are purified by’sizeexchi- sion chromatography. However, other purification techniques known in theartcanaiso beusod, including, but not limited 0, fon exchangechromatography. hydrophobic interaction ehro- ‘matography (HIC), and roverse-phase chromatography. (See eg, Maniatis eta, supra; Ausubel eta, supra; and ScopesUS 7,393,928 B2 35 (1994) Prowin Purification: Principles and Practice, Springer-Veriag New York, Ine, NY.) Prokaryotie Tnprokaryotic systems, sch as bacterial systems, any one ‘ofanimber of expression vectors may’be selected, depending ‘upon the nse intended forthe polypeptides to be express. For example, when lange quantities ofthe recombinant gelatins of the present invention, or polypeptides from which these recombinant gelatins can be derived, are needed, vec- ‘ors which direct high-level expression of fasion protein that ‘canbe realy purified may be used, Such vectors include, but not limited! to, multifunctional Ecol cloning and expression vectors such 3s BLUESCRIPT (Stratagene), inwhich the ‘encoding sequence may he ligated into the vector in frame with sequences for the amino-terminal Met and the subse- ‘quent seven resides of f-galactosidase so that yb protein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) 1. iol. Chem. 264:S508-5500): and the lke GEX (Promeya, Madison, Wis.) and pET (Invitrogen) veetors may also be used to express foreign polypeptides as {sion protein with glutathione transferase (GST). In gen- cra, stich fusion procins are soluble and ean easily be pri fed from lysed cells by a variety of methods known inthe att, or example, by adsorption to gtathione-agarose beads fle Joweal by lution in the presence of free glutathione, Proteins made in such systems may be designed to include heparin thrombin, or fietor XA protease cleavage sites so that the ‘lone polypeptide of interest can he released from the GST moiety Yeast In preferred embodiments, the present invention provides methods of producing recombinant gelatin sing a yeast ‘expression system. In preferred embodiments, uelatin is pro ‘duced dreelly from altered collagen constructs or decived from processing of collagenous polypeptides. A number of -,non-consitutve, or inducible promoters may be used ia yeast systems, (See, 8, AUD] ‘etal. supra, Chapter 13.) In some aspects, vectors containing sequences which direct DNA integration into the chromo- some are used for expression in S.cerevisiew none embodiment, the recombinant gelatins of the iwen= tion, or the polypeptides from which these gelatins can be “derived, are expressed using host cells from the yeast Saccha- romycer cerevisiae. Saccharomyces cerevisiae can be wsed ‘with any ofa large number of expression vectors available in the art, including a aumber af vectors containing constitutive ‘or inducible promoters suet asa factor, AOX, GAL I-10, and PGH. (See, Ausubel eta, supra, and Grant etal. (1987) Methods Enzymol. 153:516-544,) Commonly employed ‘expression vectors are shuttle vectors containing the origin, ‘of replication for propagation both in yeast and the ColE:1 ‘origin for E.coli, including a yeast promoter and terminator or efient transcription of the foreign gene. Veetors incor porating 2 plasmids include, but are not limite to, pWYG4 and pYES2, which have the 231 ORI-STB elements, the GALI-10, ete. In one method af the present invention, ia ‘which 2 hydroxylated product is desited, involves the co- ‘expression of a collagen post-transational enzyme, for ‘example, prolyl 4-hydroxylase. none such method, using the pWYG4 vector, the Ncol cloning siteisusedto insert the pene Tor either the tor B subunit of prolyl 4-hydroxylase, and t0 provide the ATG start eadon fr either th eof subunit Ia ‘one method, expression plasmids are sed which direct into ration into the chromesome ofthe host “The expression vector pWYG7L, which has intact 24 ORI STB, REPI and REP2, the GALT promoter, and the FLP 0 o 36 terminator, can also be used. When the co-expression of a post-ransiational enzyme, for example, prolyl 4-hydroxy~ Jase, is desired, the gene fr ether the cor subunitof prolyl ‘hydroxylase is inserted in the polylinker sith its $end at ‘Bam of Neol site, The Weetor containing the prolyl 4-hy ‘droxylase gene is trnsformed into S. cerevisiae either before ‘rater removal ofthe cell wall to produce spheroplasts that take up DNA on treatment with calcium and polyethylene alyeol orby testment of intact cells with lithium ions. Aker patively, DNA can be introduced by electroporation. Trans- Tormants ean be selected by using host yeast cells that are auxotrophic for leucine, uyptophane, urseil or histidine ‘together with selectable marker genes such 2s LEU2, TRPI, URA3, HISS or LEU2-D. Tn another prefered embodiment, the methods of produc ‘ecombiaant gelatin according to the present invention se host cols from the yeast Pichie pastors, or from other species of non-Saccharompces yeast that possess advantages in producing high yields of recombinant protein in sealed-up pracedures, Pichia expression systems include advantages of both prokaryotic (eg, E. oli) expression systems high: level expression, easy scale-up, and inexpensive growth — and eukaryotic expression systems—protein processing, Tolding, and posttransational modifications. Such expees- sion systems ean be constrcted using various methods and its available to those skilled ia the rt, for example, the PICHIA EXPRESSION kits available from Invitrogen Cor poration (San Diego, Calif) ‘There are a number of methanol responsive genes in rmethylotephic yeasts such as Pichia pastoris, oF Pichia ‘methanolica ct. the expression ofeach being controlled by ‘methanol responsive regulatory regions (also referred to as promoters) Any of such methanol responsive promoters are suitable for use in the practice of the present inveation. Examples of specific regulatory regions include the promoter or the primary alcohol oxidase pene from Pichia pastoris AOKI, the promoter forthe secondary alohol oxidase gene trom Pichia pastoris AOX2, the FLDI promoter, the promoter forthe dihydroxyaeetone synthase gene from Pichia pastoris (DAS), the promoter fo the P40 geno tc. Typically, fexpresion in Pichia pastors is obtained by the promoter trom the tightly regulated AOXT gene. (See, eg, Ellis etal. (1985) Mol. Cel. Bio. $:1111;and U.S. Pat No. 4.855.231.) Constitutive expression ean also be achieved using, e.g, the GPH promoter. Anotler yeast expression system prefered for use inthe methods of the present invention makes use of the methy- Totrophie yeast Fansemula polymorpha. This system can be used, for example, in a method of production of the present invention where high yield is desirable. Growth on methanol results in the induction of enzymes key in, such as MOX (methanol oxidase), DAS (dihydroxyacetone symthase), and FMED (Formate deydrogenise) These enzymes can const fteup 10 30-40% the total cell protein. The genes eneoding -MOX, DAS, and FMDH production are controlled by strong promoters induced by growth on methanol and repressed by growthon glucose. Any oral three of these promoters may be used to obtain high level expression of heterologous sequences in 7. poh morpha, aecording to methods known. thea. ‘none method! of de present invention, the encoding pot nucleotides are cloned into an expression vector under the ‘control ofan inducible. pormorpha promoter. secretion ‘ofthe product is desired, a polynucleotide eneoding a signal soquence for Secretion in yeast, such as MPa, i fused in frame with the ending sequence forthe polypeptides of the ‘vention. The expression vector preferably contains an sx-US 7,393,928 B2 37 ‘trophic marker gene, such as URA3 or LEU2, or any other ‘marker known in the art, which may be used to complement the deficiency of anauxotrophichost Alternatively, dominant selectable markers such as zene or blastacin may be used ‘The expression veetor is then ted to transform H. pol ‘morpha host cells using techniques known to those of ski the ar. An interesting and usefal Feature of H. polymorpha transformation isthe spontaneous integration of up 10 100 ‘copies of the expression vector into the genome, In most ‘eases, the integrated sequences form multimers exhibiting 2 head-to-tail atangement. The integrated foreign DNA. has been shown to be mitotically stable in several recombinant strains, even under non-selective conditions. This phenom- ‘enon of high copy integration further ads to the productivity potential of the system Plant ‘The present invention also contemplates the production of the recombinant gelatin of the present invention or polypeptides from which the recombinant gelatin can be derived, ia plant expression systems, including plant host cells and rans penic pants. (See, eg. Transgenic Plants: A Production Sys- tem for Industal and Pharmaceutical Proteins, Owen and 3, Galin Applied Plant Biotechnology, Chapra etal. eds, Seieace Publishers, Ine, 1999, In eases where plant expression vectors ae used, the expression of seguences may bedrivenby any of anumber ‘of promoters. For example, viral promoters such asthe 338, ‘and 19S promotes of CaMV may be used alone o ia combination with the omoga leader sequence from TMV, (Soo, ‘eg. Brisson etal, (1984) Nature 310:511-514; and Take ‘mats, N. (1987) EMBO J. 6:307-311.) Plant expression vectors and reporter genes are generally Known in the art. (See ‘eg, Gruber et al. (1993) in Methods of Plant Molecular Biology and Biotechnology; CRC Press.) Altematively plant promoters such as the small subunit of RUBISCO or best shock promoters eg, soybean hsp'7.5-F ‘orhsp17.3-1may be used, (See, eg.,Contzzi,G. etal. (1984) EMBO 1. 3:1671-1680, Broglie, R. et al. (1984) Science 224:838843; Winter, J. etal. (1991) Results Probl. Cell Di= fer 17-85-105; and Gurley et al. (1986) Mol. Cell. Biol 6:559-565,) These constricts can be inteodveed into plant cells using Ts plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, mierinjection, electroporation, pathogen-mediated transfection, particle bombardment, of any other means known in the art, such as ae described in 2 rumber of generally available reviews. (See, eg Hobbs, S. ‘or Murry, E- Ein McGraw Hill Yearbook of Science and ‘Technology (1992) MeGiraw Hill, New York. N.Y. pp. 191 196, Weissbuch and Weissbach (1988) Methods for Plant Molecular Biology, Academic Press, NY, Section VILL. pp. 421-463; and Grierson and Corey, Plant Molecular Biology, 24 Fa, Blackie, London, Ch. 7-9.) In various embodiments, the recombinant gelatin of the present invention, or polypeptides from which the present recombinant gelatin eat be derived, is produced from seed by ‘way of available seod-hased production techniques using, for ‘example, canola, com, soybeans, rice, and barley seed. In such embodiments the protein is recovered during sd ger rination/molting. In other embodiments, the protein is ‘expressed directly into the endosperm or into other parts of the plant so that the gelatin is non-extrcted, and the plant itself can serveas, for example, a dietary supplement such as source of protein. Promoters that may be us to direct the expression ofthe polynucleotides may be heterologous or non-heterologows 0 o 38 These promoters ean also be used to drive expression of antisense aucleicacidsto eedvee increase, oralte expression as desired. Other modifications may be made to increase ‘and/or maximize transcription of sequences in plant or plant fel are standard and kaon wo those inthe ar, For example, the polynucleotide sequences operably linked to a promoter ‘may finer comprise a least one factor that modifies the transcription rate ofthe encoded polypeptides, such 2, for ‘example, peptide export signal sequence, codon usage introns, polyadenylation signals, and transcription termina- tion sites. Methods of modifying nucleic acd constructs t0 ‘nerease expression levels in plants are generally known inthe fan. (See, 8. Rogers etal. (1985) J. Biol. Chem, 260:3731 Comejo etal (1993) Plant Mol Biol 23:567-568.) In engi- sneering a plant systems that affets the rate of traseription of the polynucleotides, varios factors known in the art, inci ing regulatory sequences such as positively or negatively acting sequences, enhancers and silencers, chromatin stc- tore, ee, ean be used Typical vectors usefl for expression of foreign yenes plants are well known in the ar, eluding, but not fimited 10, Vectors derived rom the taor-inducing (1) plasmid of Agrobacterium tumefaciens, These vetors are plant intgrat- ing vectors, that upon trnsionnation, integrate 2 portion of the DNA into the genome ofthe host pant (See, e.2., Rogers etal. (1987) Meth. In Enzymol. 153:253-277; Schardl etl. (1987) Gene 61:1-11; and Berger etal. (1989) Proc. Nat Acad. Sei, U.S.A B6:8402-8406,) Procedures fr transforming plant cells are available inthe an, including. for example, direct gene transfer, in vitro pro- {oplast transformation, plant virus-mediate tansformation, Jiposome-mediated transformation, microinjection, lee: ‘woporaion, lerobaererium-mediated transformation, ad ballistic patcle accoleration. (Soe, e,, Paszkowski tal (1984) EMBO J. 3:2717-2722; US. Pat, No, 4,684,611; European Application No. 0 67 $83: US. Pat. Nos. 4.407 956; 4,536,475; Crossway etal (1986) Biotechniques 4320. 334; Riggs et al. (1986) Proc. Nal. Acad. Sei USA 83:5602- ‘5606; Hinchee etal. (1988) Biowechnology 6:915-921; and US. Pat. No. 4.945.050.) Standard methods forthe transfor ‘mation of rice, wheal, com, somghum, and baeley are described inthear. (See, e.,Christou etal. (1992) Trends in Biotechnology 10:239; Casas tal, (1993) Proc, Nat'l. Acad. Sci. USA 9011212; Wan etal. (1994) Plant Physiol. 10:37 and Lee et al. (1991) Proe, Natl Acad, Sei, USA 88: 6389.) ‘Wheat can be transformed by techniques similar to those employed for transforming com or ree. (See, eg, Fromm et al. (1990) Bio’Technology 8:833; and Gordon-Kamm et al supra.) Additional methods that may be used to generate plants or plant cells that can express the present recombinant gelatins, fr polypeptides from which these recombinant gelatin can be derived, are well-established in he art. (See, g., U.S. Pa. [Nos. 5,959,091; 5,859,347; 5,763,241; 5,689,122; 5,593,874; 5,495,071; $424,412; 5,362,865: and 5,229,112.) ‘The present invention firter providesa method of produc- ‘ng polypeptides by providing biomass from plants or plant cells which are comprised of at least one polynucleotide sequence encoding 2 recombinant gelatin, of a polypeptide ‘rom which recombinant gelatin canbe derived, wherein such polynucleotide sequence is operably’ linked to a promoter to effect the expression of the polypeptide. Ina further embodi- ‘ment, the method additionally comprises co-expression of at Ieqst one polynucleotide sequence encoding an enzyme that catalyzes. a postranslational ‘modification, or subunit thereof, wherein such polynucleotide sequence is operablyUS 7,393,928 B2 39 aked fo promoter. In these methods, the recombinant ela tins or collagenous polypeptides are extracted fom the bio- Fungi Filamentous Fungi may also be used to produce the polypeptides ofthe instant invention. Vectors for expressing ‘and/or secreting recombinant proteins ia filamentous fungi ‘are well known ia the at, and one of skill a the at could, using methods and products available in dhe art, use these vectors in te presently recited methods. (See, eg, U'S, Pt No. 834,191.) Insect Insee cell systemsallow forthe polypeptides ofthe present mention to be produced in large quantities. In one such system, Autographa californica nuclear polyhedeosis virus (AeNPV) is used as a vector to express foreign genes in, for ‘example, Spodoptera frugipera ces oe in Trickoplusia lar vae, Sequences encoding the gelatns or gelatin precursor of the present invention may be cloned into non-essential regions ofthe virus, for example, the polyhedron gene, and placed undercontrol ofan AeNPV promoter for example, the Polyhedron promoter. Successful insertion of a coding Sequence will result in inactivation ofthe polyhedron gene ‘and production of non-ocelded recombinant vints (.e, vis lacking the proteinacenus cost encoded by the polyhedron ene), These recombinant viruses are then used to infest ‘Spodoptera frugipenda cells or Trichoplusia larvae in whieh, Iynueleotides encoding the gelatns or gelatin precursors ‘expressed. (See, e.. Pngelhant,F. K. tl. (1994) Proc. Nat, Acad, Sei, 91:3224-3227; Smith etal. (1983) J. Virol 46:584; and U'S. Pat. No. 4.215.081). Further examples of this expression system may be found in, eg, Ausubel et al (1995), supa ‘Recombinant production ofthe polypeptides ofthe present invention can be achieved in insect eels, for example, by Jngeetion of baculovimas vectors containing the appropriate polynucleotide sequences, including those encoding. any Post-translational enzymes that might be necessary. Bate Joviruses are very efficient expression vectors forthe large- scale prodition of various recombinant proteins in insect cells. Various methods known inthe at ean be employed to ‘consirvt expression vectors containing a sequence encoding, ‘gelatin or gelatin precursor of the present invention and the ‘appropriate. ranscriptionalanslational control signals (See, eg. Luckow et al (1989) Virology 170:31-39: and rueawald, 8, and J, Heitz (1993) Baculovirus Expression \ector System: Pracedures & Methods Manual, Pharmingen, San Diego, Calif) Animal ‘The present invention provides methods of expressing the recombinant gelatins of the present invention, or polypep- fides from which the recombinant gelatins of the present Jnvention can be derived, in animal systems, Such systems jnchide mammalian and non-vertebrate host cells an rans- teenie animals. In mammalian bos ells, a sumer of expres ‘in systems may be utilized. In cases where an adenovirus is lused a8 an expression vector, sequences encoding the polypeptides ofthe preset invention may be ligated into an adenovirus transerptionauslation complex consisting of the late promoter and tripartite leader sequence. This chi erie gene may then be inserted inthe wlenovinis genome by ‘of in vivo recombination Insertion into a non-essential E] or E3 region of the viral penome may be used t obtain viable vinis which is eapable of expressing the polypeptides ‘of the present invention in infected host cells. (See, e. 0 o 40 4. and Shenk, . (1984) Proc. Natl, Acad. Sei 3659.) Alternatively, the vaccinia 75 K promoter may be used. (See, eg Mackett etal. (1982) Proc. Nat Acad. Sei, USA 79:7415-7419 (1982); Mackett etal (1984), J Virol. 49:887-864; and Panicalie al, (1982) Proe. Nat! Sci. USA 79:4027-4031.) In addition, various transcription enhancers known in the art, such as the Rous sa- ‘com virus (RSV) enhancer, may be used to inerease expression in, for example, mammalian hostels, ‘Seni Forest vs sa preferred expression system asthe viru has abroad host range such that infection of mammalian cll nes willbe possible. Infection of mammalian host cells, for example, baby hamster kidney (BHK) cols and Chinese Jhamstercary (CHO) cells, sing sucha viral vector canyiekd very high recombinant expression levels. More specifically it is contemplated that Semliki Forest virus ean be used in a ‘wide range of hosts, asthe system is not hased on chrome- sonal integration, and therefore willbe aguick way ofobiain- ing modifications of the recombinaat gelatine in studies simed at ideniying structure-funetion relationships and test- ing the effects of various hybrid molecules. Methods for cconstreting Semliki Forest virus veetors for expression of ‘exogenois proteins in mammalian host cells are know inthe ar and are described in, for example, Olkkonen et al, (1994) Methods Cell Biol 43:3-53, Additionally, CHO cells deficient in dihydrofolate reduc tase (dhir) can be transfected with an expression plasmid ‘containing a dlr gene and the desired polynucleotide, Selec- ‘ion of CHO cells resistant to increasing concentrations of rmethoirexate will undergo gene amplification, providing higher expression lovels ofthe desired rocombinant protein, ‘asknowa in the ar. "Transgenic animal systems may also be used o express the eeombinant gelatns of th present invention or the polypeptides from whieh these recombinant gelatins can be derive Such systems can be constructed, for example, in mammals by operably linking an encoding polypeptide fo a promoter and other required or optional regulatory sequences capable fof effecting expression in mammary” glands, Likewise, required or optional post-traslational enzymes that effect post-ranslational modifications, may be produced simul ‘ously inthe target cells employing suitable expression s tems. Methods of using transgenic animals to recombinantly produce proteins are keowa in the ar. (See, eg. US. Pa. Nos. 4,736,866; 5824838; 5.487.992; and 5,614,396; and co-pending US. application Ser. No. O8/087.202.) Uses of Gelatin Gelatin appear inthe manufacture or as a component of various pharmaceutical and medical products and devices, It js estimated that about 85% of pharmaceutical produets eon- ‘ain hovine-derived materials in some form, ineluding the y used. in various products, for example, pharmaceutical stabilizers, plasma extenders, sponges, hard and soft gelatin capsules, suppositories, etc Gelatn’s film-forming capabilities are employed in various {ilm coating systems designed specifically for pharmaceutical ‘ral solid dosage forms. including controlled release capsules and tablets, and other numerous pharmaceutical products in \whieh gelatin serves asa coating intended 10 improve ease of ‘administration and delivery, et. Gelatin appears 3s stabi Jizer in various forms, for example, in the pharmaceutical industry, e., in drugs and vaccines, in food and boverage products and processes, in industrial applications, eg. eon- rete stabilization, and as a stabilizer in various laborstory solutions, eg, various cell preparationsUS 7,393,928 B2 4 Gelatin in various edible forms has long been used in the ood and beverage industries, Gelatin is used widely in vari- ‘ous confectionery and dessert prodoets, particularly in puddings, frostings, cream fillings, and dairy and frozen prod ucts, Gelatin serves as an emulsifier and thickener in warous ‘whipped toppings, as well asin soups and sauces. Gelatin is used as a floeculating agent in clariiving and fining various beverages, including wines and fruit juices. Gelatin is used in various ow and rece fat products, such asmayonnaiseand salad dressings, as a thickener and stabilizes, and appears ‘elsewhere as fat substitute, Gelatin is also widely used in mieroencapsulation of favorings, colors, and vitamins. Gela- tin can also be used as a protein supplement in various high ‘energy and nutritional beverages and foods, such as those prevalent in the weight-loss and athletic industries. As fil- Former, gelatin is used in coating fits, meats, del items, and in various confectionery products, including candies and um ete In the cosmetics industry, gelatin appears in varity of hair care and skin care products. Gelatin is used asa thickener and bodying. agent in a. number of shampoos, mavsses, ‘reams, lotions, face masks, lipsticks, manicuring solutions ‘and products, ox other cosmetic devices and applications ‘Gelatin s also used in the cosmetics industry in microencapsulation and packaging of various products. Gelatin is used in a wide range of industrial applications For example, gelatin is widely used asa glue and adhesive in ‘various manufacturing processes, Gelatin ean be wed in varie ‘ous adhesive and gluing formulations, such asin the manu ‘cture of remoistenable gummed paper packaging types, ‘wood gluing, paper bonding of various grades of box bowls and papers, and in various applications which provide adhe- five surfaces which can he reactivated by remoistening, Gelatin serves asa light-sensitive coating in various elec tronie deviees and is used as a photoresist base in various photolithographic processes, far example, in color television ‘and video camera manufeturing. Ia temiconductor manu facturing, gelatin is used in constructing lead frames and in the coating of various semiconductor elements. Gelatin is used in various printing processes and in the manufacturing of special quality papers, such as that used in bond and stock cerifieates, ee ‘Use of gelatin in photographic applications is long-established, Gelatin is used asa career for various active compo= nents in photographic safutions, including solutions used in X-ray and photographic film development. Gelatin, long used in various photocngraving techaigies, is also included as @ ‘component of various types of film, and is heavily used in silver halide chemistry in various layers of film and paper products, Silver gelatin film appears in the form of microfiche ‘iim an in other forms of information storage. Gelatin used asa selfsealing element of various films, ot. latin has also been a valuable substance for use invari ‘ous laboratory applications. Por example, gelatin can be used in various cel culture applications, providing a suitable surface for eell attachment and growth, eg. as a coating for plates asks, microbeads, or other substrates, or providing & suitable protein source in growth media, Hydrolyzed or low ‘gl srength gelatin is used as biological buffer in various processes, for example, in coating and blocking solutions ‘ised in assays such as enzyme-linked immunosorbent assays (ELISAs) and other immunoassays. Gelatin s also a compo- rent in varios gels sed for biochemical and electrophoretic analysis, including enzymogrephy pels a Pharmaceutical The presen invention also contemplates the use of recom binant gelatin in various pharmaceutical and medical app cations. In particular, n one embodiment, the present invention provides fora pharmaceutical composition comprising recombinant gelatin, In prefered embodiment, the recom- ‘binant gelatin is derived from human sources. The present recombinant gelatins offer an advantage previously una able in the art that of using gelatins derived from native ‘human collagen sequence, thus reducing the risk of immuno- sdenecity tothe gelatin material, In addition, a the present gelatins are produced recombinanlly ina controlled environ- ‘meat, rsks of infectivity, from agents such as TSEs oF from pathogens and endotoxins intraduved during processing, are ‘minimized Endotoxin levels of commercial materials typically ange from about 1.010 1.5 EUimg of gelatin. (See, eg, Schagaer, Hand G, von Jagow (1987) Anal. Biochem. 166-368-379; Friberger, Petal. (1987) Prog. Clin, Biol. Res. 231:149-169.) Inthe methods ofthe present invention, the endotoxin levels can be reduced by two to three orders of magnitude, (See Example 8.) The present invention thus provides, in one embodiment, «recombinant gelatin derived from human sources that is virtually endotoxin-free In adaition to providing a gelatin material without the fectivity issues associated with ani- ‘malderived materials, the present invention allows for a reproducible source of consistent product. Specifically, the present gelatns ean be presented as a homogeneous mintare ofidentical molecules, The physical charactristis desire in 4 particular medical application ean he specifically intro- {duced and achieved consistently. The present inventions thus fhle to provide a reliable and consistent product will mini- 1 variability associated with the availability and use of ‘current gelatin products In specific embodiments, the recombinant gelatin of the present invention can be used in the manufature of capsules, including hard shell or hard capsules, typically produced from gelatin solutions, and softshell or soft eapstles yp call made from gelatin fils. I specific embodiments ofthe present invention, a hard gel apse comprising recombinant selatin anda soft gel capsule comprising recombinant gelatin fare provided, as are methods for manifacturing these capsules. The themoreversbility of gelatin is a property exploited in a number of applications, for example, in the ‘manufacture of such gel capsules and tablets, Gelatin ean be ‘ated, molded, oF shaped as appropriate, and can Be used 10 {forma eapsule or tablet coating that has unique properties at homeostatic temperatures. A selected gelatin can begin to ‘melt at mouth temperature, easing swallowing, and become liquid at internal body temperature, such as within the stomach, In one emboxtiment, the present invention provides recombinant gelatins with the dissolution rates of commer cially available capsules and coatings. In another embodi- ‘ment, the present invention provides recombinant gelatins ‘with improved resileney, appropriate for use in capsules and tableting In ceriain applications, such as the manufacture of gel capsules, the Britleness nd hardness developed by gel ‘over time isan important parameter that ean limit the shel Jifeand usefulness of curently available animal-source gele- tins, The ability o maintain viscosity overtime would be a valuable asset, especially for manufacturer of gelatin-containing products, who curently buy gelatin in sizable lt in ‘order fo maintain consisteney of manufactured products. Fur thermore, some mantfacturing processes, stich as the man tacture of hard gel capsules, cueretly require a blend of 0 oUS 7,393,928 B2 43 elatin ypes, eg. of type A andl type B pelatins, in order v0 produce a material withthe desire properties as the use of ‘ypeB gelatinalone results, for example, ina hard gel capsule that isto brite for maniaaetare and use ‘The recombinant gelatins of the present invention ae of s ‘greater purity and are better chareterized than curently available materials. Thus, te present gelatins can provide & Sable material, and one more reproducible and predictable in its behavior. Furthermore, using the methods ofthe present jnvention one could engineer a recombinant gelatin that pos- sessed the structural features of both iypes of gelatin in & ingle molecule of in a wellebaraeterized mixture of mol- cules. "The recombinant gelatin ofthe present invention can also be used asa stabilize in various pharmaceutical products, for ‘example, in drugs or vaccines. (See, eg. co-pending, eo monly-owned U.S. patent application Ser- No. 09/710,269, ‘entitled “Recombinant Gelatine in Vaccines,” fled 10 Nov 2000, incorporated herein by reference in its entirety.) There- Tor, ia one embodiment, the present invention provides @ stabilizing agent comprising recombinant gelatin, whorein the stabilizer is suitable for use in pharmaceutical ppl tions. Ina preferred embodiment, the recombinant gelatin is recombinant human gelatin, Different regions of various collagens are associated with various activities, for example, various regions of type II] collagen have been associated with active sites involved inthe ‘loting cased, Therefore, in one embodiment, the present invention contemplate the se of polynucleotides eneoding recombinant gelatns that contain specific active regions ofa particular collagen or of particular collagens. Sach poly- nueleotides ean be used in a variety of ways, for example, in ticroamrays. Such polynvcleotdes could thus be ws as & ‘diagnostic tool to identify altered links of mRNA polynvcle~ blides comesponding to collagenous domains of interest in 3 sample. The encoded polypeptides could be used in various methods of sereening for drugs or compounds that could inhibit or enhanee the activity andor expression associated ‘with particular collagenous domains. The present gelatin can also be used in encapsulation, ‘nchuding miceneapsulaton, and in tableting, supp ries, and various medical emulsions. The present invention also contemplates the wse of the recombinant gelatin provided herein in medical sponges, eg. hemostatic sponges, et in woud treatment and in varius surgical applications, eg as sponges used to prevent leakage after port removal in fetos- ‘copy and other procedures. Therefore, in one aspect, the present invention comprises a sponge comprising recombi ‘ant gelatin, wherein the sponges suitable for usein medical procedures. In a prefered embodiment, the recombinant felatin is recombinant human gelatin, ‘The recombinant geatins of the present invention ean be designed w possess specific physidl properties suitable for tase in particular applications. The present invention provides methods for varying characterises suchas molecular Weight, ‘gol srength, and pH of the final gelatin formulation to pro- ‘duce gelatins with specific properties as desired, and to thus ‘meet customer's specifications to degree unatainable with ‘currently avilable materials. Moreover, such formulations allow the customer to explore refinements of existing pro- ‘esses and formulations, as well as to develop new applica tions, fr the present recombinant gelatns “The moleeniae weight distributions of commercially available animal-lerived soluble gelatin, such as those used in ormulation of vaccines, range from about 0 to 30 KD and from about 010 60 KD. (See Fxamples 7 and 9.) The present invention provides for 8 method of producing recombinant 0 o 44 latins, under suitable hydrolysis conditions, that results in recombinant human gelatns with molecular weight Aistrbutions which corespond with the commercially avi able welatins, and can be used forthe same purposes. Addi- ‘ional, the reseat invention provides meiods for producing gelatine with « narrower molecular weight distribation, or example, about 10 0 30 kDa, or about 30 to $0 kDa, not available from commercial materials ‘The recombinant gelatin of the present nveation, and compositions thereof, can also be used in various surpical proce- ‘dues, including in biodegradable conduits for directing and supporting nerve regeneration, in colloidal volume replace- ‘ment in major surgeries, in gelatin sponge plugs used to seal various port sites, such as atheterizaton sites and other inci- sions or wounds, and in polyester grafts as an infection- resistant sealant. (See, e.g, Migilehe,N.. etal. (1999) Past Afi Med. J.76(7):400-406, Beyeret al. (1997) Br. J. Anaesth 7(1):4-80; Luks et al. (1999) Am. J. Obstet. Gynecol. 181 (4):995.996; and Farooq etal. (1999) J Surg. Res. 87(1)57- 61) “The present pharmaceutical compositions can be admin tered to a subject for treatment of various joint conditions including arts, athosis, and other conditions related to the degeneration of cartilage and joint flexibility. In pre- {ered embodiment, the rocombinant gelatin contains a mod- fed amino acid sequence which possesses higher concentr- tions of arginine, hydroxyproline, and hydroxylysine, and other amino acids elated 1 the prodvetion of collagens and proteoglycans in cartilage (See, eg, Oesser etal (1999) J ute, 129(10):1891-1895,) Microspheres synthesized with the goats ofthe present invention are also contemplate. Sueh microencapsulated particles ean be used, for example, in directed delivery of therapeutic proteins or small mol- cules, providing 4 noainflammatary and biocompatible delivery system. (See, eg, Brown et a, (1998) Arthritis Rheum, 41:2185-2195,). Inanother aspect, the present invention contemplates oral administration of the recombinant ageletins of the present invention toalleviate disease aetivty in ‘heumatoidarlritis.(Arboreliu etal (1999) Rheumatol Int 18:120-135,) In ingested pharmaceutical proets it might be desirable to provide recombinant gelatin having stability aginst degradationia the aeidic environment the stomach, aut et ‘Techniques for encapsulation, and various formulations and devg delivery systems, are available in the art and are escribed in numerous sources. (See, ez, Gennaro. Re (1990) Remingion’s Pharmaceutical Sciences, 18° ed, Mack Publishing Co., Easton Pa.) The most effective and conve- sient route of administration and the most appropriate foe lation fora particular simation can be readily determined by ‘methods known in the art Suitable routes of administration may, for example, include oral, rectal, transmucosa, or intestinal administration And parenteral delivery including intramuscular, subeutane- ‘ous, intramedullary injetions, as Well as intrathecal, diroct intaventricula, intravenous, intaperitoneal, intranasal, oF intraocular injections, Vaccines, for example, can be deliv ered intravenous, nasa or oral and ean take the form of ive ‘attenuated, subunit, monovalent, divalent, cevatent vaccines, te, Formulations for enterie release, etc, are also eontem- plated, The composition may be administered ina Toca rather {hana systemic manner. The present invention also provides ‘pharmaceutical eomposition comprising recombinant gela- tinwhercin the composition is sitable for delivery as. spray, {or Hingual or nasal deliveryUS 7,393,928 B2 45 Food Inthe food industy, gelatin’ s physical properties and pure protein composition make it stable for use in a varity of ‘ways, including as a eomponeat of various edible products ‘and aulrtional supplements, Gelli can bea food product in its own right, proving a cathohydrate-ree, pure protein source. In addition, gelatin’ physical and strctoral characteristics are use in various food preparation and packaging applications, For example, gelatin is used as a gelling and thickening agent, as an emulsifier and foaming agent; to prevent cling or protei-liguid separation; for “eel,” orto Improve consistency and texture; w retain moisture: and in ‘edhesion and packing, for example, as an edible Hn ible gelatin can serves a particularly valuable source of pre protein. Therefor, in one aspect, the present invention provides a protein supplement comprising recombinant gelatin. The gelatin of the present invention ean be produced with, or example, specific and desired amounts of essential amino acids. The present invention provides for the production of ‘various edible gelatins, wheter in gel, leaf, or powder for ith characteristies optimal fora particular application orend prod ‘The present invention provides for recombinant yelatin products comprising different ratios of amino acid residues ‘Typically, gelatin contains most ofthe umino acids essential for humans, including for example, lysine, arginine, leucine, ‘and isoleucine. In one embodiment, the present invention provides recombinant gelatin comprising the specifi ratios ‘of amino acids desired. For example, pelatin used in foods Jnended to supplement an athlete's diet might comprise higher levels of residues such a lysine, which isbeneicial © ‘muscle growth, and arginine, which, a a precursor to eret- in, is involved in the energy metabolism of muscle eels. ‘Geiatin ean serve to enhanes the nutritional value of foods in Renerl by completing and increasing the aminoacid composition of other protein sources, for example, meats and dairy products Gelatin has minimal or bland taste, and can thus serve as 3 palatable and nutrional food supplement. Hydrolyzed gels tin forexample, is used asa substitute for more concentrated solutions of carbohydrates in desserts and candies and in ‘ther calorie foods, reducing the calorie content. Gelatin ean ‘ao serve ws a source of protein i foods with high nutritional valuo, foe example, low-calorie foods produced in the diet industry or high-enemy foods. In addition to serving as @ protein source, gelatin can serve asa earbohyerat-free car Fer filler substance in, for example, spray or dred instant ood proves and fhworings, or asa elanfying and fining agent in, for example, wines and juices, The ability of gelatin to impart desirable characteristics, including, for example, texture, color, and clarity, is highly ‘valued, The textureof sich products depends toa largedegree ‘onthe typos of ingredients used, formulation variables, and how the productsare processed and handled. In confectionery applications, for example, gelatin appears in a varity of peed products, such as pasties and popular gummy prodlets. Gelatin is used a8 a gelling agent, providing textures ranging from soft and elastic 1 short and hard. The texture and mouthfeel of the finished product is dependent on the bloom strength, concentration, and formulation of gelatin used, In addition, gelatin’ s colloidal properties provide substrate for colors andl dyes allowing the desired opacity oF ‘larity, aswell as color of the end product. Therefor, in one ‘aspect, the present invention allows forthe use ofa gelatin that provides the desired textural properties, biliance, and ‘larity, inthe manufacture of gelled confectionery products, 0 o 46 Inanother aspect, anappropriate gelatin s selected which has relatively low viscosity high viscosity can produce unde- Simable“tiling’ of the depositing syrup during manufature, causing defective products. Generally speaking. the higher the bloom value of the gelatin, the harder the product ‘becomes, so that by inereasing the gelatin eonten, the prod- vet becomes hander and chewier in texture. A property of yelatin widely exploited in, for example, the production of aerated confectionary prods is its ability to pradce and support aYoam, and to promote rapid sting at the aieTiquid interface by forminga film around entrapped air bubbles. Aerated products constitute a lange family of cone fectionery products, including marshmallows, frostings, now gals, and cookieand wafer fillings. The degree of seration and Setting time required fora particular product depends on the {ype and grade of gelatin used, ogether withthe concenin- ‘ion of gelatin in the final product, Altering the type and proportion of gelatin used ean vary the texture of aerated products. For example, getatins with high bloom values, oF fel strength, produce a shorter chess, whereas gelatine With lower bloom values provide a moce elastic texture. Gelatin serves a numberof functions in the manufactureof fit chews, and other sugae-pulled confectionery product ‘types, suchas toflees andearamels, which contain fats and are slightly aerated. For example, gelatin assists in the emul cation of fats, improving dispersion and stability: provides Sesirable texture and chesvines, as well as foaming ability land contrbutesto the shelf ofthe inal produc, sch as by ‘controling suerose crystallization. Gelatias with a Bloom of lout 150 to 200 to are typically used in these products at ‘sage levels of 05-1.5% wi. Therefore, in one aspect, the present invention contemplates recombinant gelatin wth a Taloom of about 150 40200 for use in edible products Gelatin provides cohesive texture in eream pastes, which contain both soli and liguidl phases consisting of powdered ‘sugar and fas dispersed in a sugar syrup. Gelatin acts a8 2 binder to prevent # enumbly texture and to inhibit cracking. Gelatn's binding properties are also wilized in lozenges and compressed tablets In products suchas licorice, gelatin, often ‘combined with an agent such as wheat flor, acts asa binder, greatly improving moisture eteation, and preventing erack- ing and crumbling during: manufieture. Gelatin also helps prevent confectionary products, such as, for example, licorice, rom drying out in storage, improving product shit Tie ‘The present invention thus provides, in one embodiment, binding agent comprising recombinant gelatin, which bind- ‘ng agent can be a component of edible procicts. The present invention further provides @ moisturizing agent comprising recombinant gelatin, which moisturizing agent is suitable for se in edible products. Gelled products are avilable in various forms, including ready-to-eat products, dry blended powdered mixtures, oF tablets in which the sugar, gelatin, acids, lavoring, and col- coring have been dissolved and gelled. Gelatn's ability 10 orm elastic-extured thermo-reversible gels with melting points around 25-38” C. is exploited in seh uses. The final ‘extre, rigidity, and setting rate ofthese gelling products are controlled by the concentration and physical properties ofthe aelatn, most particulary, bloom strength and viscosity levels, In the production of gelatin desserts, the use of a lower ‘concentration of a higher-gmde gelatin to produce a gelled product of @ panicular rigidity would provide advantages, ‘including economie advantage, as well as improved clarity tnd color development, compared to the use of a higher jon of 3 lower strength gelatin. Therefore, ‘embodiment, the present invention provides a gelling ageUS 7,393,928 B2 47 ‘comprising recombinaat gelatin, wherein the pelling agen is suitable for use in an edible product ‘Gelatin is often used inthe manufacture of various dairy products, such as icecream, yogurt, and puddings, in which 2 particular texture and mouth feel is desire; in particular, ‘gelatin provides a smooth, even-extured consistency and ‘reamy mouth fel. Gelatin is used in combination with other hydrocolloids a thickener and stabilizer in low fat mayon- naise and salad dressings. ‘With the expansive growth inthe number and desirability ‘of low and no-ft dairy produets, gelatin ean make an out Sanding contribution othe product texture, body, and mouth {et of a finished procuet. With its ft-like melting character Jstes, a gelatin having a melting point of around 25-35° C. provides the desirable sensory properties, or “meltn-the- ‘mouth’ characteristics, thus simolting the texture ofthe full: fit product, Ina health-conscious society, gelatin is well-suited for use asa stabilizer in low or reduced fat and non-fat youu prod- Uuels, adding to the body and mouth-feel, and creating. @ Smooth dicate. and ereamy texture inthe absonee of fat Additionally, gelatin stabilizes these products by preventing syeresis, or the separation of whey proteins In this regard zeltin products function to form a gel network which binds ‘water, preventing exudation and separation ofthe whey pro teins, tus helping produet shelP-ife. Gelatin i also used the manufacture of thickened creams, in which te gelling and emulsifying properties of gelatin are used to inerease ‘ream viscosity. Gelatin also has widespread use in sour ‘ream, soft cheese produits, and acidic milk desserts, suchas ccheesccakes, nd in flavored milk-based desserts, stich a5 rmousses,chiffons and soules, The cream viscosity can be ‘varied es desired by altering the concentration and gellin properties of the gelatin used. Typical gelatin levels for su! tses rage from 0.2-0.8% wi, although higher or lower gel strengths could be desired in various products. The present invention provides a stabilizing agent comprising recombi nant gelatin “There is increasing demand inthe food and health ins tries for reduced fat or fat-free products. Gelatin’s dietetic properties, including its ability to provide protein in the absence of fat, make it useful inthe weight-loss industey, as well as in products designed for patents, comvaescens, and individuals with special dietary sensitivities or needs. Gela- tins protein content adds carbohydrate free aurtional value. Inaxlition to its nuteitonal value, gelatin is highly digestible ‘and can thus be administered in liquid foods that are easily ‘absorbed, Pure gelatin contains no fats, sugars, purines, oF ‘cholesterol. Gelatin’s physical properties, protein content, and lack of strong taste make it a preferable fat substitute in ‘many products. Gelatin is widely used 2s an emulsion stab lize in, for example, products such a low-fat butters and margarines. As a thickening and binding age, gelatin cua replace in whole or in part the fat content in various food products. For example, gelatin can replace highly calorie Binders suchas crear, butter, and other dairy fats oe yolks: ‘and other starchy products. In addition, geltin's moisture retaining qualities are helpful in binding large amounts of wate, allowing for greater postprandial satisfaction and fll “The sensory or mouth fel of gelatin is critical, a6 many fat-ffoe of reivced fat products seek to mimie as closely’ as possible the mouth fect, as well asthe taste, of fats, By using ‘gzolatn in a low-fat formulation, it is possible to achieve a Texture comparable to a full-ft produet, thereby achieving & lower calorie content while preserving prefered extoreand ‘mouth-feel. The amount of gelatin used is dependent on the 0 o 48 percentage offi ifany, contained inthe finished product. For Instance, ata fat content of 60%, 0.5% wi gelatin i use hile at lower fat levels of 25%, approximately 3.5% wit gelatin is used to maintain product integrity and sensory appeal. Gelatin peodicod according to the preset invention ‘an possess a melting-point similar to that ofthe food prod ‘eis in which itis include or, preferably, the body or mouth temperate of humans, resulting in melting of gelatin at eating temperatures and a comrespondingly rich mouth-fel In addition, gelatn's bland taste will not interfere with the dTavorings Of a particular food produet. Finally, gelatin is highly digestible ‘Using gelatin asa fat substitute thus allows fora reduction in ealories without a corresponding reduction in texture and richness and without coeresponding negative effeetson taste and digestibility. The present inveotion, in one aspect, pro- Vides fat substitute comprising recombinant. gelatin, ‘wherein the fat substitute i intended for use in edible prod vats In preferred embodiment, the recombinant gelatin has ‘melting point of from about 25 to about 35°C. Gelatin improves the appearance and slicing character ties of various canned and preserved foods, including meats stich as cooked ham, by penetrating and filling any cavities in the tissue. In canned meat products, gelatin serves to absorb the juices that are released during the retoring process, oproving he slicing properties and giving a pleasing appearance 1o the product. In these instances, a gelatin should be ‘elected that has fow caleium content as precipitation of calcium phosphate fom the phosphates inthe meat juices can ‘eeu In canning applications, such as canned seafood, a gelatin with a high gel strong is used to withstand the thermal tatment applied during the sterilization process. Depending on the exten of sterilization and the get strength selected, gelatin levels usually range from 0.5-5.0% wiv. Gelatin also serves a a binder and gelling agent in canned seafoods and meats and ina variety ofjelled (apie) produes Gelatin finds application for satsage coatings, where it is cused as an adhesive agent in binding spices tothe surface of products soch as salami. The sausage is dip-coated in 2 concentration solution of gelatin that typically has a high ‘loom and high viscosity giving the gelatin time to set and ‘nhibiting run from the predict surface. Such coatings are also used, for example, in the manufacture of soybean and ‘ther substitute meat products, and in the evating of various Tits, meats, and delicatessen items. In one aspect, the present invention provides an edible coating comprising recombinant gelatin, Gelatin is also used in mieroencapsulation of various fa vors, colors, and other additives, and of vitamins. Specifically contemplated are various recombinant gelatins that can be used as stabilizing azens, thickening azets ‘ilm-forming agents, binding agents, edible coatings, gelling ‘agen, protein supplements, emulsifying agents, microen- capsulants for colors, favors, and vitamins ee, and can be used in various food supplements, including nuteitional and dict supplements, and fat substitutes. Inone embodiment, the gelatin ofthe present invention is used inthe processing or packaging of, or as component i, foods prepared for cone sumers with Kosher, Hala, vegetarian, or other diets that restrict the ingestion of food containing specific animal- source products In adlton to being used in edible products intended for ‘human consumption, gelatins are used as binding agen in the manufaeture of bars and pellets in pot foods, snacks, and chevwables. Ia addition to the siuctural advantages gelatin offers in these products, gelatin's high protein cantent can contvibute positive effects such alleviating symptoms ofUS 7,393,928 B2 49 degenerative diseases of the animal skeletal system, as wells improving pelt growth and texture, Photographic In another aspect, the present invention comprises & photographic composition comprising recombinant gelatin Pef= ‘erably, the recombinant gelatin is partially hydroxylated, ‘Gelatin sa key component of various photographic processes ‘and produets, eluding, forexample films and paper Gelatin js used as a binder in light-sensitive products, whet its gel- setting and film-forming properties make for cleat, uniform and durable coatings which ean involve multiplecoatings in & Single application, Gelatin as a binding agent ereates and provides the uniform consistency solidification, or cohesion ‘desired, Gelatin also stabilizes couple and dye emulsions in ‘color photographie products Gelatin is indispensable in photographic coatings includ ing silver halide emulsion layers, top coat or surface layers, interlayers, and back-coats, The chemical and colloidal properties of gelatin enable precise precipitation and chemi- ‘al ripening of photographic silver halide emulsions. Some ‘emulsifying fluids use non-gelling fish gelatin, which may remain Hiqud in solutions at concentrations as high as 40%, and st temperatures as low as 20° C In one embodiment, the recombinant gelatin has a low ‘molecular weight and a low setting temperature. In snthee ‘embodiment, the recombinant gelatin has alow seting point, but a higher molecular weight than available in curentnon- selling piscine-derived gelatins or in animal-derived gelatin hydrolysates The recombinant gelatin of the present invention ean be used in various photographic applications, for example, for the support of silver halides on hoth film and paper. In one ‘embodiment, the recombinant gelatin hss a setting tempera ture of between 15° and 25° C. The recombinant gelatin can bespray-dried and offeredasa low density, cold water soluble powder or film, and is ths advantageous for use in various technical applications, for example, photoresist systems, The present gelatin can also be used in gelatin filters. The present vention contemplates photographic gelatin products cus- ‘om-designed to meet the exacting properties ofeach particu- ‘need, as well as methods for making such gelatine, Other ‘The recombinant gelatins of the present invention offer various technical advantages over commercially available gelatin due to its more particular and integrated chemical ‘make-up, and the corresponding consistency in its physical properties. The recombinant gelatin of the present invention ‘can thus be used in technical applications which currently Jnvolveextracted gelatin. Porexanpe, the preset gelatin cua be used ina variety of industrial processes, including, but not Timited to paper sizing and photogravure, colotype, seven Printing processes, mieroencapsulated dyes, copy transfer papers and olher papers and boards costed with gelatin through the formation of a coacervate complex with gum arabic, Gelatins ofthe present invention can also be uscd in ‘electroplating toensuresmooth deposition andasa protective ‘colloid in some polymerization reactions, and a a coating oF film-forming agent in semiconductor manufacture In another embodiment, the present gelatin is used as 2 binder for special quality papers, inclnding tock eeifcates, bank notes, ete, The present gelatin further serves asa bond- ‘ng agent for use in match paste, providing a lower density ‘andl more even combustion for matches, a well as fastening ‘of abrasive particles on a canvas or paper hacking to produce abrasive papers 0 o 50 The distinctive properties of gelatin, inluding its ability 0 serveas a protective colloid, and to alter is electrical change ‘with changes in pH, combine (© make gelatin a material stable for use in microencapsulation. Gelatin and ts derivatives can thus be used in a variety of mieroencapsulation vices and techniques, for example, inthe microcncapsula- ‘ion of inks for earbon-itee paper fragrances for advertising and sample manufacture; chemicals use in multi-component adhesives; and vitamins and nutritional supplements. The ‘microencapsulation capabilites of gelatin and its derivatives fare also useful in the manufietre of packaging materials, ‘including packaging allowing minimal permeability for oxy. sgn, aromas, ancl water vapor, Gelatin is thus widely used in exible packaging, such as packaging for food, phamacen- sicals, and other sensitive products The adhesive effet and reduetion of suriee tension peo- vided by pelatns render them useful in ea fertilizers. Due to the stability and slow degradation of the amin acids of gela- ‘in the prevsely adjusted nitrogen concentration provided by the ferilizeris thus maintainedandmadeavailable ofa longer period of tine. Gating are also useful as @ biologically ‘ogradable binding agent inthe mantafactare of feilizer pel lets ue to its amino acid composition, gelatine can serve as complex sources of nitrogen usefil, for example in the synthesis of penicillin by Penicillium chrysogemum, as well as orexample, nthe manufactoreof various starter cultures and antibiotics. (See, eg Leoaharsberge, etal. (1993) J Bio- ‘wchn! 30:299-313,) ‘Te recombinant gelatin of the present invention can be ‘sed in various laboratory applications, in whieh the repro- ‘debility and uniformity of the recombinant gelstns ofthe present invention will be greatly valued, minimizi ‘unwanted variability in laboratony processes and compos ‘ions. For example, the present recombinant geatins can be used in various tissue culture applications, providing a su able protein source in growth media, and, in some applications, providing a cell growth matrix o scaffolding, or other surlace for ell sttachment and growth. The present invention ‘also provides a cell preservation fomulation comprising recombinant gelatin, She formulation could, Forexanple, be ‘sed to preservea preparation of platelet cells, protecting the solution until administration and use. The present invention contemplates biological buffers comprising hydrolyzed or Jow gel strength recombinant gelatns, seh as various blocking ‘nd coating solutions. In further embodiments, the present invention provides reproiible recombinant geatins {or use in variows gels used for biochemical and clectro- phoretie analysis, including enzymography gels. ‘The present invention also encompasses microcarier beads eosted with recombinant gelatin, Such microcariers, ‘sed, eg. in mammalian cell culture, provide a growth sur face for attchment-dependent cells. Polysaccharide and polysiyrene beads, for example, can be coated with the recombinant gelains of the present invention to provide a stale surfice for cell tachment and growth. In one ‘embodiment, the microcarier beads othe present vention are coated with specific recombinant gelatins containing aetve collagenous domains capable of inducing differentia tion and growth of particular ell, Different regions of various collagens are associated with various activites, for example, various regions of type IT collagen have heen associated with ative sites involved inthe clotting cascade. Therefore, in one embodiment, the present Jnvention contemplates the wse of polynucleotides encoding recombinant gelatins that contain specific active egions of aUS 7,393,928 B2 51 particular collagen or of particular collagens. Such poly- ruecleotides can be used in a variety of ways, for example, in ricreaerays, ‘Recombinant gelatins, polypeptides, and polynvcleotides ‘encoding the recombinant gelatins of the present invention ‘ean he tsed in novel microarmy technologies and sereening methodologies. Collagen fibrils and immobilized collagen bind strongly to platelets, a platelets have multiple binding site for collagen that encompass several collagen molecules polymerized to cach other. The interaction of platelets with ‘collagen trough their collagen reveptors results in aetvation ‘of the platelets and subsequent fomnation of platelet aggre ates Recombinant gelatins consisting of biologically active regions of collagen typeI, fr example, can be prepared as microibers that consist of uniformity, purty, and reprode- ‘bility unattainable with current collagen and gelatin sources “Microfibers derived from the present recombinant gelatine ‘ean be preseated on substrates, eg, arrays or chips, used t0 fereen Tor compounds that prevent platelet aggregation through interaction with, e, type Il eollagen, or any other ‘eil-forming collagen. ‘Chemical compounds, small mol- ‘cules, peptides, or ther biological molecules (uch as ant bodies ea be screened for their ability to prevent, reduce, oF slow the process of clot formation or platelet agaregation, mediated by platelet nteraetions with specific regions withi collagen fiber, such as, for example, RGD sequences. Addi ‘ionaly, microarrays would also be useful for examination of the interaction of different types of integrins with various regions of collagens and gelatin microsfibers. Microfibers produced from recombinant gelatine from any ofthe fire orming collagens, e collagen typeI type IL type IL type \ortype XI, cou bese in sercening for collagen-induced platelet aguseyation antagonists "Akko contemplated are microarays of polynucleotides ‘encoding recombinant gelatins or fragments thereof. Such Ietoaerays are sefil in sereening for and isolation of vari- ‘nis of eollagen- oF geltin-encading polyauclestides, DNA or RNA, and in determining differential levels of ‘expression in, for example, normal vs. diseased tissue. Tn another embodiment, the present invention provides purified recombinant human gelatins for use in the dfferen- tition of progenitor cells, for tissue rogencraton therapies, and for tissue engineering. Components of the extracellulae ‘matrix ae involved in the regulation of ell proliferation and 59 10 m1 ‘00> seoumncR: 1 geacetoteg agangagaga ggetgaaget gatetgceta ataceaagaa & oo “210 589 10 m2 ‘00> seoumncR: 2 gestetctes ageagagaga goctsagect gzagcteaga gaccesctge © 2 eto» #89 30 ms <2to> #40 30 m -too> sepumce: 6US 7,393,928 B2 67 ~continued 68 soo suqumce: 7 210 su9 1D ¥0 8 too suqumice: © egctetagat cattanggey egeeagatte accyetgtte ceettasy 210 S09 10 ¥0 9 400» sgrimce: ° egctetagat cattatetct egeetettge tecagagas 210 su 1 ¥0 10 400» sgrimice: 10 srgccostas teaggcragt gtgatggsat cocetggace tanagutact gottaat ‘210 s49 1 ¥o 11 4000 sngrimce: 11 21> sto 10 80 12 soo» swgumice: 12 ‘210 st 10 80 13, ‘21> 549 10 80 14 21> 50 1D No 15US 7,393,928 B2 69 ~continued 70 too sequmier: 18 cay Pro Mat ly Pap Sex Oly Ose Reg Oy Uns Bro IY Pro Pkg hy ‘a Pro ely Reo Gln Oly Phe Gin Gly Exo Bro Gly Olu fre Gly Goa ro hy Rag Ger Ghy Pro et Ohy Pro Aig Gly Pro Pip aly io Hse ly lye dan Oly Aap Rap Oly 6B4 Ae Oby Lys PEO IY Axo Po hy <210> #49 1 ¥0 16 soo» sxgumce: 16US 7,393,928 B2 n ~continued nD 210 S40 1 8017 $00» segumiee: 17 ay Pro Mat ly Pap Sex Oly Bee Reg Oy Las Bro IY Pro Pkg hy ay neg Oy Pro Pro Gly Pro Gin Oly Ala Ag Gly Inu Pro Gly The ‘a Gly teu Pro Oly Mot bye Gly Hike Arg Gly Phe Ser Gly tow Rep cy fro Gin Gly Pro Gly Gly ro Pro Gly Pro Lys Gly Aen Ser Gly ‘ctu Pro Gly Ala Pro Gly Ser Lys Gly Agp The Gly Ala Lys aly ale3 US 7,393,928 B2 ~continued 4 aw aa ay Pre aay Pro cay tye aap Pro ala aly ein aay teu ay au an 210» sn9 10 80 Leu Pro a1y, aap aly Lye iy ou Ate uw Arg any Pro Gty Pro aay var Pre 400» sngrimce: 210» 549 10 8019 ‘soo segumcr: 19 ae tye me aay oy ate aa aly ay asp hy tou the Gay ser Pre ony sex fo bro Gy Pro Aaa ely Gln Rep tm Oy Pro Pro Gay Pro Ata chy any Pie Re ay See Pro hy Phe fo Pip Gly Ou Rta ay tye Bro eu oxy Ae fro Gay Pro Ser chyUS 7,393,928 B2 15 ~continued 16 210» 580 10 No 20 too» sequimiee: 20 uy ma ny teu Pao Gly Ala Lye Oly teu Me GIy Sex Pro Gly Sex ro Oly Po Aap Ghy tye The Gly Bro Pro Oy Pro Ala Gly GIs Rep cay mg Pro Gly Pso Pro Gly Bee Bro Oly A Arg Ghy Gls ALA Sly Val noe cay Be Peo ly Pup Lye Gly Ala Ae Gy Glu fro Gly Lye ‘Ag cay cau Arg oly Van Pro Ghy Pro Pro Gly Ate Val Gly Pro Ale 2102 549 10 #0 21 Yak Met ly He P50 Gly Peo Lys Oly Ala Ale Gly Glu fro Bly LysUS 7,393,928 B2 7 ~continued 8 ‘ig Arg Gly G14 Aig Oly Phe Bxo Gly GLH Arg Gy Val Gin Gly Pee Pro Gly Pro Ala Gly Pro Arg Gly Ala Ran Gly Ale Pro Gly Aen Aap iy Bex Pto Oly We Rap hy Val Reg hy Una The GIy Pro Tie hy Pro Bro Oy Pro Rig Gly Ala Bro Oly Rp Wye <210> #49 10 ¥0 22 oo» segues: 22 Fro Ghy Pro lye Gly Aap Arg Sly Aap Ria Gly fre lye Shy Ala Rep79 US 7,393,928 B2 ~continued 80 2a tap cay Gin Pro Oty Ale yg ety top Ala hy Poe Peo cay bro Te ony Aen van ely Ser ma Gy Bro Bro Ghy Ala tye ay any ro Gay Aap ALa oly Ata fo Maa Gay Pro Ata Gy Pro Fee fo Oly Ale Uys Gay Ata Reg chy hy Pe Pre Gay Ria Aas Oly Ag ‘a ory ou tye gly Ser Pre iy the Pro Gly Pre Gin Gly op cay sex Pro ‘210 s49 10 ¥0 23 ‘soo sxgumcr: 23, ay Ma tap chy Pro Na Oly Bg PHO ag chy ean arg Oty Ya Ve ty arg Gly Glu Gin Gly pro Ala Gly ser pro Gly Phe Gin Gly Leu Pro ‘arg oly Phe ro Gly Glu Arg Gly Val Gin Gly Pro Pro aly Pro ALA 210» S40 1 80 2481 US 7,393,928 B2 ~continued 82 soo» sequmic: ass ay aay Pre eg ay op mia eae ace ay ag ay ay Mp oy 210» sn0 10 ¥0 oo» sugrice: pep ala ay ay ap ay ay ay ay ay ay ay ay ay ay aay tye aay ay aap ay ay ay ay as ay ay ay ay ayUS 7,393,928 B2 ~continued 84 Pro ay aay Pre tye cis Pro ay ay ay ia Pro Gty ‘ee cry Phe ay Aen ana, aay bye aay, vat aiy Pre 210» sto 10 80 26 oo» segues: 26 ay ap tye ay ay ay ay ay as ay ay ay as ay tye Ghy dep Ra va aly ay Pre as ay ay ay ay byeUS 7,393,928 B2 85 ~continued 86 ro ia Gly Peo Ris Oly io Oxo Oly Oo Be Gly don VOL Oy Ra ro Gly Ala We Ghy Ala Atg Gly Sex Ala ly fro Pxo Gly Ala The cry Pro Pro ly bso Pro Gly Bro ALA Oly Olu ye Ghy Sot Bro Oly 2a Pop Gly Bro Ma Oly Ala fro Gly The Bro Gly Pro Gin Gly Te aa chy Gap Arg Oly Val Vad Gay teu Bro Gly Gls ig Gly Gla AaB ly Me Peo Oly Leu Pro Oly fre Sex hy Gia Bk Gay Lys Gln hy op Arg chy Glu The Gly Pro Ala Gly Pro Pro Giy Ala Pro Gty Ala is ely ala pro Gly Uys Asp aly Leu Aon Gly Leu Pro Gly Po toes sequimiee: 2787 US 7,393,928 B2 ~continued 88 aay Pre aep aly ay aa ag aay ag ay ae ay ay ay ay oy ‘210 sto 10 80 28 ay oy ay ay ay oop ay ne ay ay aw Pro aay say sex tye ay arg Pro aly ye aay Ser aay Pre can, an arg @ay Pro any Ale ay ata Pro) aly any uve au ver aly Pre Gly Ala ay ne ate au arg aly bye ony Aen aay ata PoUS 7,393,928 B2 89 ~continued 90 Pro Gin Gly Pro Arg Gly Aep tye Gly Gia the Gly Glu Gin Gly Aap ‘og Ghy Tae Lye Oy ke Arg Gly She Sex Gay Leu Gin Gly Pro Pro Ary Pro Pte Oly Sex Pro ly 634 Gin hy Pro Sex GIy AYA Ser Gly ip Ma Gly Peo Rey Gly Do Ose Oly Sor ABA Gly Ala Pro Oy Lys ‘ep chy teu han Gly Ueu Pro Ghy Pro Tae Gly Pro Pro Gly Bre Reg cay ig Me Gly Aap Ala Gly Bro Val Oy BHo Pro Gy Bre Pro Gly 4210» 549 10 ¥0 29 4000 sugrimice: 20 fg Guy Aop tye Giy Giu Thr Giy Giu Gin Giy Aop Arg Gly The Lye 210 st 10 80 30 arg Gly Glu ain Gly pro Ala Gly ser pro Gly Phe Gin Gly Leu ro 210» 580 1 No 22mn US 7,393,928 B2 ~continued 92 swqumnce. 21 cay mis Gy Ala ip Gay Peo arg Gly Glu Gln Gly Pre ay 9 eg Gy the Bro oly Oty mo Aig Gly Pre Pre ro Gly hap Le Oty op Ala ciy Ala Pro c1y Gin Gry Met Pro cty cia Guy Aap Arg Gly Aap Ala pro cay as tye ciy cis ‘589 10 wo 32, ay ay Pro aay aay ser Pro aay aay aa Pre tye ay ay aay tye ay ‘589 mo wo 33 ny Pro ata oly ona Phe Gin Gly Lew Pre bro aay atu ain oy any Aa Arg cay ons teu Pro Gly Pre bye fap chy Sex Pro Oxy93 US 7,393,928 B2 ~continued of na ay ay ew da eg Pro ay ay ae ay ay we ag oy ay we we ay ay ap ay ay Pre tye Pro ay uy tp arg aly aay me cay Ale ae ay ay ay aay ag ay aay ois Pre aby We a Vel Gly Pro ALA Pro Gly Pre Ser aty ay vat ain aly Pre aa Pro chy Ron Rep fo ay ger Gin atyUS 7,393,928 B2 95 96 ~continued ay tay Ala Pro Gly Ala Pro Gly Ala Pro Giy Pro aay tye sex cry Rep au Pro ala 2g oly clu Thr oy ay Pro Val @1y Pro val aly Ala arg Guy Pre as ay ay Pre ag Gly Bop. bye Guy cia the aay au ein @1y aap ie tye aay Hie Ag cay Phe ser aay teu cin Guy Pro Pre vai aly ay Pre ag ay aay Pre ‘What is claimed is 1. A recombinant gelatin comprising. the amino acid sequence of SEQ ID NO30, 2, An encapsulant comprising the recombinant getatn o ha 3. stabilizing agent comprising the rovombinant gelatin of claim t 4. film-forming azent comprising the recombinant gets tin of elain I a ‘3. An emulsifier comprising the recombinant gelatin of ‘lain 1 6.A thickening agent comprising the recombinant pelatin of claim f 5 7.A colloidal agent comprising the recombinant gelatin of * ‘lain 8. A hard gel capsule comprising the recombinant pelatin of claim 1 9. soft gel capsule comprising the recombinant gelatin of 5, ‘lui 1 10.A plasma expander comprising the recombinant gelatin of cai 11. A colloidal volume replacement material compsising the recombinaat gelatin of claim 1 12. A medical sponge comprising the recombinant gelatin of claim f 4 o 13. pharmaceutical stabilizer comprising the recombi- ‘ant gelatin of claim 1 14, The pharmaceutical stabilizer of claim 13, wherein the ‘hamaceutical stabilizer isa vaccine stabilizer. 18, A microcarier comprising the recombinant welatn of claim 1 16, An edible composition comprising the recombinant gelatin ofelaim t 17. A protein supplement comprising the recombinant gelatin of elaim the recombinant gelatin of 19, A nutritional supplement c aclatin of claim f 20, An edible coating comprising the recombinant gelatin ofelaim I 21, A photographie composition comprising the recombi ‘ant gelatin of claim 1 12. A cosmetic composition comprising the recombinant gelatin of claim t 23. An industrial composition comprising the recombinant aclatin of claim L 24, Acellculture composition comprising the recombinant gelatin of lai f ising the recombinant

3 Recombinant Gelatins

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3 Recombinant Gelatins

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