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1040070138_TERM PAPER

1040070138_TERM PAPER

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LOVELY PROFESSIONAL UNIVERSITY

TERM PAPER OF CONCEPTS IN BIOTECHNOLOGY ON PRODUCTION OF RECOMBINANT PROTEIN IN EUKARYOTES

SUBMITTED TO, HARSH KUMAR. SUBMITTED BY, R109B44

ACKNOWLEDGEMENT
I here by acknowledge that this term paper “PRODUCTION OF RECOMBINANT PROTEINS IN EUKARYOTES” has been made under the guidance of Mr. Harsh Kumar. Yet there is always room for improvement, I invite you for the improvement of this term paper.

CONTENTS
1. INTRODUCTON 2. ADVANCES IN THE PRODUCTION OF HUMAN THEREUPEUTIC PROTEINS IN YEAST AND FILAMENTOUS FUNGI 3. RECOMBINANT PROTEIN PRODUCTION IN YEAST - EXPRESSION VECTORS

4. 5. 6. 7. 8. 9.

FACTORS AFFECTING THE LEVEL OF RECOMBINANT PROTEIN PRODUCTION PRODUCTION OF RECOMBINANT PROTEINS IN OTHER MICROPRGANISM PRODUCTION OF HUMAN FACTOR V||| ADVANTAGES OF USING ANIMAL CELL LINES FOR RECOMBINANT PROTEIN PRODUCTION OVER PRODUCTION OF RECOMBINANT PROTEIN USE OF STRONG CONSTITUTIVE PROMOTER INCLUSION OF AN INTRON INCLUSION OF POLYDENYLATION SITE REMOVAL OF UNNECESSARY AND UNTRANSLATED SEQUENCES OPTIMIZATION OF TRANSGENE SEQUENCE FOR EFFICIENT TRANSLATION INCORPORATION OF TARGETTING SIGNAL RESEARCH ON RECOMBINANT PROTEIN REFERENCE

INTRODUCTION
Recombinant DNA Technology involves the isolation and cloning of genes of interest, production of the necessary gene construts using appropriate enzymes and then transfer and expression of these genes into an appropriate host organism. This approach is also

called genetic engineering. This technique has been used to achieve the following two broad objectives: (1) Production of recombinant proteins (2) Modification of the organism’s metabolic pattern for the production of new, modified or more quantity of metabolities. In this term paper I am going to discuss about recombinant protein.

RECOMBINANT PROTEINS
Proteins produced by genes transferred by genetic engineering into selected host cells are called recombinant proteins since their production is based on recombinant DNA technology. Recombinant proteins form an important component of biopharmaceuticals, i.e. , biotechnology products having pharmaceutical applications. A large number of recombinant proteins are being produced in mammalian cell cultures, some of which, viz., human growth hormone, tissue plasminogen activator, erythropoietin and blood clotting factor V|||, are already in therapeutic use. The host cells used for large scale production of the various recombinant proteins is as follows: Chinese hamster ovary line, baby hamster kidney line BHK, mouse mammary tumors line C127, mouse my eloma cell lines and mammalian cell lines. The use of cell line has been made possible by the following developments in cell culture technology: 1 Development of culture systems permitting large scale culture of animal cells at high densities 2 Development of media, which minimize or even obviate the use of serum. These developments have enhanced the yields and reduced the costs of same products obtained from genetically engineered bacteria and yeast. Even when an animal cell line produces a desired protein on its own, expression vectors and cloned genes are still used to maximize yields by using a much stronger promoter than the one present with the endogenous gene. Often the promoter used in expression vectors comes from a virus like SV40. The cost of production from mammalian cell lines increases remarkably due to the strict implementation of rigorous quality control measures to ensure that the product is free from viruses that infect mammals. Insect cell cultures provide an attractive alternative host system for recombinant protein production. The expression vector is based on baculoviruses, which do not attack vertebrates. The product of polyhedron gene of baculoviruses accumulates in the insect cell nuclei as large inclusion bodies towards the end of infection cycle; polyhedron makeup over 50% of the total cell protein. The transgene is inserted in the place of polyhedron gene, but all the signal sequences of the transcription unit are retained. The recombinant protein is produced at the same level as polyhedron . Several mammalian proteins have been produced in insect cell cultures, but unfortunately the proteins are not glycosylated correctly; this remains the chief limitation of this system.

Advances in the production of human therapeutic proteins in yeasts and filamentous fungi
Yeast and fungal protein expression systems are used for the production of many industrially relevant enzymes, and are widely used by the research community to produce proteins that cannot be actively expressed in Escherichia coli or require glycosylation for proper folding and biological activity. However, for the production of therapeutic glycoproteins intended for use in humans, yeasts have been less useful because of their inability to modify proteins with human glycosylation structures. Yeast glycosylation is of the high-mannose type, which confers a short in vivo half-life to the protein and may render it less efficacious or even immunogenic. Several ways of humanizing yeast-derived glycoproteins have been tried, including enzymatically modifying proteins in vitro and modulating host glycosylation pathways in vivo. Recent advances in the glycoengineering of yeasts and the expression of therapeutic glycoproteins in humanized yeasts have shown significant promise, and are challenging the current dominance of therapeutic protein production based on mammalian cell culture.

RECOMBINANT PROTEIN PRODUCTION IN YEAST
Yeast is a unicellular eukaryote and can be easily grown in continuous cultures like bacteria At, present yeast is the most popular alternative to E. coli as a host organism as (1) It exhibits relatively high levels of recombinant protein production (2) It is considered as a safe organism for production of such proteins that are to be used in medicine or in food (3) The genetics and biochemistry of yeast are well documented (4) Eukaryotic recombinant proteins are folded properly and disulphide bonds are regularly formed. One of the serious limitations of yeast is (1)The improper glycosylation of the proteins as it often hyperglycosylates the recombinant protein; such proteins can be immunogenic in animals. It can be reduced by using appropriate mutant strains of yeast. (2) Yeast lacks an efficient system of protein secretion in the medium, which increases the cost of protein recovery and purification. (3) Sometimes, codon bias may result in poor trasgene in unable to efficiently excise heterologus introns; therefore foreign gene has to be used as its cDNA version.

Expression Vectors

Yeast expression vectors are based on the yeast cloning vectors. These vectors carry a suitable yeast promoter, e .g., GAL, PH05 or CUPI promoter, and the transcription termination sequence as well since animal promoters and termination signals are very poorly recognized in yeast. GAL promoter comes from the gene that encodes galactose epimerase, an enzyme involved in galactose metabolism; this promoter is induced by galactose. Similarly, PH05 promoter is induced by removal of phosphate from the culture medium, while CUPI is induced by copper. In addition, several constitutive promoters, e.g. , PGK(phosphoglycerate kinase), ADHI (alcohol dehydrogenase) and GAPDH(glyceraldehyde-3-phosphate dehydrogenase),have also been used.

Factors Affecting the Level of Recombinant Protein Production.
Several factors affect the level of recombinant proteins produced by yeast cells; some of these are listed below. (1) Number of copies per cell of the expression vector. (2) Overall stability of the expression vector; smaller vectors are more stable than the larger ones. (3) Promoter strength; stronger the promoter, higher is the expression. (4) Stability of the heterologus mRNA; this is one of the major rate determining factors. It should be noted that mRNA half-life can not be accurately predicted from its base sequence; therefore, it has to be empirically determined. (5) The presence of correct transcription termination signal at the 3 –end of mRNA; it enhances mRNA stability. (6) The 5-region of mRNA should be optimized for translation in yeast. For example, the presence of an upstream AUG in the mRNA leader sequence effectively inhibits translation initiation in yeast, while it is immaterial in mammals. (7) Recombinant protein degradation in the cells and during downstream processing; the problem can be minimized by using protease deficient strains as host. (8) Extracellular secretion protects recombinant protein from proteolysis.Secretion can be achieved by adding the appropriate short hydrophobic signal sequence at the N-terminal end of the recombinant protein.Signal sequence from yeast genes encoding invertase, phosphatase, killer toxin, etc. have been used. In this strategy, the transgene is inserted downstream of the signal sequence and there is translational fusion.

PRODUCTION OF RECOMBINT PROTEIN IN OTHER MICRO-ORGANISM
(1) Pichia pastoris is another species of yeast; it produces recombinant proteins up to 30% of the total cell proteins. Its glycosylation abilities are very similar to those of animal cells, although the sugar structures do show trivial differences from those in animal proteins. Expression vectors use the AOX promoter from the gene

that encodes alcohol oxidase; this promoter is induced by methanol. But P. pastoris sometimes degrades recombinant proteins; this can, however, be controlled by using special growth media. (2) Filamentous fungus Aspergillus nidulans has good glycosylation properties and secretes proteins into the growth medium. Expression vectors for A. nidulans usually use glucoamylase promoter, which is induced by starch. (3) Trichoderma reesei has the same desirable features as A. nidulans. Expression vector for T. reesei use the cellobiohydrolase promoter, which is induced by cellulose. (4) Other yeasts that have been used for recombinant protein production include Hansenula polymorpha, Yarrowia lipolytica and Kluveromyces lactis, the last of these can be grown on wastes from the food industry.

PRODUCTION OF HUMAN FACTOR V|||
Human factor V||| plays a central role in blood clotting. The most common form of haemophilia results from an inability to synthesize factor V|||. Factor V||| for the treatment of this form of haemophilia was earlier obtained from human blood provided by donors using a complex purification procedure. More importantly, there was always the risk that factor V||| preparations were contaminated by dangerous viruses, and there have been cases of transmission of hepatitis and AIDS viruses to haemophiliacs via factor V||| injections. This risk is indeed associated with any protein purified from human blood. Recombinant factor V||| is now produced in a Chinese hamster cell line and, more recently, in pigs. The gene encoding factor V||| is very large having 26 exon. The mRNA encodes a 2,351 amino acids long polypeptide, which undergoes a complex series of post-translation processing events and glycosylation at several sites. The functional factor is a dimeric protein; its large subunit is derived from the N-terminal end, and the small subunit comes from the C-terminal end of the original polypeptide. The two subunit are held together by 17 disulphide bonds,. E. coli is a very poor choice for a host to produce a protein like factor V|||; therefore, it was produced in Chinese hamster cell lines as follows. (1) Initially, the complete cDNA sequence of factor V||| gene was expressed, but protein yields were very low. It was reasoned that post-translational processing was not efficient in the Chinese hamster cell lines. (2) In the next step, the two regions of factor C||| cDNA that encode the large and small subunits were isolated and each was integrated into a separate expression vector under the control of Ag promoter. Ag promoter is a hybrid between chicken Actin and rabbit-globin promoter sequences. Both the vectors were introduced into a Chinese hamster cell line; the yield of protein was 10-times the yield when the complete cDNA was used, and the protein was identical to native factor V|||.

(3) The most current strategy is the production of factor V||| in pig. The complete cDNA has been driven by the promoter of whey acidic protein gene of pig; the factor V||| is now produced in pig mammary gland and is secreted in the milk. This recombinant factor V||| appears to be identical to the native human proteun. This appears account should give an idea of the considerations and difficulties involved in the production of recombinant proteins of pharmaceutical value and the approaches that can be used to resolve them.

Advantages of using Animal Cell Lines for Recombinant Protein production
The use of animal cells for the production of recombinant proteins is preferable to the use of microorganisms for the following reasons. (1) The signals for synthesis, processing and secretion of the concerned proteins are better recognized in animal cells than in microbes. (2) The protein products are readily secreted into the medium, which makes their recovery much easier. (3) The patterens of folding and disulphide bridge formation in the recombinant proteins are similar to those of the natural proteins. (4) The multimeric proteins are also assembled correctly. (5) There is correct glycosylation of proteins, which is a problem even in hosts like yeast.

Overproduction of Recombinant proteins
High level expression of transgenes in cultured mammalian cells is used for the production of therapeutic recombinant proteins. Expression in mammalian cells is particularly desirable when characteristic mammalian glycosylation of proteins is necessary for their therapeutic application. The following factors contribute to a high level expression. (1) Use of a stronger enhancer/ promoter, e.g., SV40 enhaner/early promoter, etc. (2) Lack of secondary structure within the 5’-untranslated region of the Mrna (3) Lack of an initiation codon in the 5’-untranslated region, i.e., before the correct initiation codon. (4) The sequence around the initiation codon should confirm to Kozok’s rule. Of these rules, the most important is the presence of a purine at the -3 position and a G at the +4 position . The A of the AUG initiation codon is counted as +2 and so on. (5) Absence of AU-rich sequences in the 3’-untranslated region of mRNA since this sequence reduces mRNA stability. The various strategies for maximizing transgene expression are briefly described in the following sections.

Use of Strong Constitutive Promoter
The use of strong promoters is a very attractive and useful strategy for enhancing transgene expression. In case of viral vectors the transgenes, the transgenes are usually driven by very strong promoters, e.g., polyhedron promoter, p7.5 promoter, EI promoter, etc. Certain viruses contain strong promoters and enhances; some of these have been used to drive transgenes. For example, SV40 early promoter and enhancer, the Rous sarcoma virus long-terminal repeat promot and enhancer, and the human cytomegalovirus promoter are the most commonly used elements to drive transgenes in mammalian cells.

Inclusion of an Intron
The presence of an intron in a eukaryotic expression unit usually enhances transgene expression. Therefore, most mammalian expression vectors in current use contain a heterologus intron, e.g., the SV40 small t-antigen intron or the human growth-hormone intron. Modified hybrd introns that match the consensus splice donor and acceptor-site sequences have also been designed and used. The preence of an intron is very important in construts that are to be expressed in transgenic animals. But introns may not be used in some expression vector sytems, e.g., vaccinia virus vectors.

Inclusion of a Polydenylation site
The polydenylation site provides the signal for generation of a defined 3’-end to the mRNA molecules, and for addition of a poly (A) tail at the 3’-end of mRNA. Poly (A) tail consists of several hundred a residue added to the 3’-end of mRNA, after transcription, by the enzyme poly (A) polymerase. Poly (A) tail is necessary for the transport of mRNA from nucleus into the cytoplasm, and it also enhances mRNA stability. When the expression system lacks a poly (A) site, the transgene expression may decrease up to 90%. In mammalian expression vector, the poly (A) sites from the SV40 early transcription unit or the mouse gene are often used.

Removal of Unnecessary and Untranslated Sequences
(1) Eukaryotic mRNA contain untranslated regions of variable le Some other 5’-UTRs codons contain sequences that affect mRNA stability, e.g., AU-rich sequences reduce Mrna stability, and such sequences have been discovered in some 5’UTRs, In addition , the 5’-and 3’-UTRs may be rich in secondary structure, which prevent efficient translation. In view of the above, the 5’-and 3’-UTR sequences are generally removed from transgene construts to maximize their expression and the production of recombinant at their 5’-as well as 3’-ends. These UTRs can influence the level of gene expression in a number of different ways as follows.

(2) Some 5’-UTRs contain AUG codons upstream of the authentic translation initiation codon; often such additional AUG codons interfere with translation initiation. Proteins.

Optimization of Transgene Sequence for Efficient Translation
For optimum translation efficiency, the sequence around the translation initiation site should be 5’-CCRCCAUGG-3’, the kozak’s consensus sequence, or as close to it as possible. In this sequence, the purine at-3 position and the Gat position +4 are of the greatest importance. The further, different organisms prefer to use different codons for the some amino acids. Therefore, if a transgene is taken from another organism, it may contain codons that are not commonly used in the host organism. In such a case the efficiency of translation will decrease; this may also lead to truncation of the transgene in such a manner that the codons not preferred in the host are replaced by those that are commonly used, but the amino acids sequence of the encoded protein remains unchanged; this is called codon optimization.

Incorporation of Targetting Signal.
Often eukaryotic proteins are modified after translation to become functional. For example, many proteins of therapeutic value require authentic glycosylation patterns for their correct function as well as for preventing an immune responses in the patients, Specific types of post-translational modifications occur in particular cellular compartments.Therefore, it is necessary to target the recombinant proteins to that specific compartment of the cell where the given modification will be effected. For example, the proteins to that specific compartment of the cell where the given modification will be effected. For example, the proteins that are to be glycosylated, must be targeted to the secretary pathways using a signal peptide. Many mammalian expression vectors contain sequences that encode specific signal peptide sequences. For example, the Invitrogen vectorpSec.Tag2 contains a signal sequence from the murine immunoglobin light-chain signal peptide; the signal peptide encoded by ttisb sequence targets the protein to the secretary pathway with a high efficiency. In addition, the C-terminus of the recombinant protein obtained by using this vector is a fusion of two different epitope tags, which facilitate protein purification. Several high level expression systems are available. When a large scale production system is envisaged, the choice of a particular system will depend on the cost of culture system, need for stable or transient transfection, and the possible need for inducible expression. For therapeutic applications, a stable, permanentlyexpression cell line consistently yields products of reproducible quality. In case of transient expression, the scale of transfection step becomes important. It may be pointed out that the maximum expression attainable may not always be required. Inducible gene expression can be achieved by using metallothionein promoter. In this case, transcription is induced by metal ions like cadmium and zinc. Alternatively, a promoter achieved by steroid/thyroid hormone/retinoic acid may be used. For example, mouse mammary tumour virus (MMTV) LTRhas a complex promoter region that is activated by steroid hormone.

RESEARCH ON RECOMBINANT PROTEIN
The Article Cloning and Characterization of Multgenesi Encoding the Immunodominant 30-Kilodalton Major Outer Membrane Proteins of Ehrlichia canis and Application of the Recombinant Protein for Serodiagnosis Send in September 1998 to theDepartment of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210-1093 It was Received at 2 March 1998/Returned for modification 7 April 1998/Accepted 16 June 1998

ABSTRACT
A 30-kDa major outer membrane protein of Ehrlichia canis, the agent of canine ehrlichiosis, is the major antigen recognized by both naturally and experimentally infected dog sera. The protein cross-reacts with a serum against a recombinant 28-kDa protein (rP28), one of the outer membrane proteins of a gene (omp-1) family of Ehrlichia chaffeensis. Two DNA fragments of E. canis were amplified by PCR with two primer pairs based on the sequences of E. chaffeensis omp-1 genes, cloned, and sequenced. Each fragment contained a partial 30-kDa protein gene of E. canis. Genomic Southern blot analysis with the partial gene probes revealed the presence of multiple copies of these genes in the E. canis genome. Three copies of the entire gene (p30, p30-1, and p30a) were cloned and sequenced from the E. canis genomic DNA. The open reading frames of the two copies (p30 and p30-1) were tandemly arranged with an intergenic space. The three copies were similar but not identical and contained a semivariable region and three hypervariable regions in the protein molecules. The following genes homologous to three E. canis 30-kDa protein genes and the E. chaffeensis omp-1 family were identified in the closely related rickettsiae: wsp from Wolbachia sp., p44 from the agent of human granulocytic ehrlichiosis, msp-2 and msp-4 from Ana plasma marginale, and map-1 from Cowdria ruminantium. Phylogenetic analysis among the three E. canis 30-kDa proteins and the major surface proteins of the rickettsiae revealed that these proteins are divided into four clusters and the two E. canis 30-kDa proteins are closely related but that the third 30-kDa protein is not. The p30 gene was expressed as a fusion protein, and the antibody to the recombinant protein (rP30) was raised in a mouse. The antibody reacted with rP30 and a 30-kDa protein of purified E. canis. Twenty-nine indirect fluorescent antibodies (IFA)-positive dog plasma specimens strongly recognized the rP30 of E. canis. To evaluate whether the rP30 is a suitable antigen for serodiagnosis of canine ehrlichiosis, the immunoreactions between rP30 and the whole purified E. canis antigen were compared in the dot immunoblot assay. Dot reactions of both antigens with IFA-positive dog plasma specimens were clearly distinguishable by the naked eye from those with IFA-negative plasma specimens. By densitometry with a total of 42 IFA-positive and -negative plasma specimens, both antigens produced results similar in sensitivity and specificity. These findings suggest that the rP30 antigen provides a simple, consistent, and rapid serodiagnosis for canine ehrlichiosis. Cloning of multigenes encoding the 30kDa major outer membrane proteins of E. canis will greatly facilitate understanding

pathogenesis and immunologic study of canine ehrlichosis and provide a useful tool for phylogenetic analysis.

REFERENCE

1. Essentials of Biotechnology-By B.D. Singh 2. Biotechnology By-R.C. Dubey 3. From Web Sites-

www.wikipedia.com www.google.com

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