Protein Sources Choice of protein source:  A prerequisite to the isolation, characterization and/or utilization of any protein is the identification

of a suitable protein source.  In a few cases the desired protein may be unique to a specific species or be produced by a very restricted member of species; e.g., the gonadotrophic hormone pregnant mare serum gonadotrophin (PMSG) is found in only equids. Under such circumstances, the choice of protein source is already made.  In most cases, however, the protein of interest will be produced by a range of species, providing a choice of source. The purpose for which the protein is required will also influence this choice.  If the protein is to be used for an applied industrial purpose, choice must be made very carefully. Recombinant versus non-recombinant production  Low natural expression levels has rendered difficult the isolation, study and application of a range of proteins from native sources. Such difficulties have been overcome with the advent of recombinant DNA technology. Now-adays, in principle, the gene or cDNA coding for any protein (of known or unknown function) can be isolated and inserted into an appropriated expression system; and a very large number of proteins are now produced by recombinant means.

Start from here Microorganisms as a source of proteins: Many proteins of industrial interests are obtained from (non-recombinant) microbial sources. The majority are synthesized by a limited number of microorganisms which are classified as a GRAS (Generally Recognized As Safe). GRAS listed microbes are: • Non-pathogenic
1

• •

Non-toxic and Generally should not produce antibiotics

GRAS microorganisms include bacteria such as • • •
• •

Bacillus subtilis Bacillus amyloliquefaciens Various other bacilli Lactobacilli and Streptomyces spp.

• •

Aspergillus Penicillium Mucor and Rhizopus

Yeast such as Saccharomyces GRAS listed fungi include members of cerevisiae are also recognized as safe.

2

.. Microorganisms represent an attractive source of protein as a) They can be cultured in large quantities over a relatively short time period by established method of fermentation. b) They can produce an abundant regular supply of desired protein product c) Microbial proteins are often more stable than analogous proteins obtained from plant or animal sources.
d) Microbes can be subjected to genetic manipulation more readily than animals

or plants. Types of proteins: • • Extracellular Intracellular

Extracellular protein products: Many industrially significant proteins obtained by methods of fermentation are secreted by the producing microorganism directly into the culture medium. Advantages:  Such extracellular protein production greatly simplifies subsequent downstream processing as there is no requirement to disrupt the microbial cells in order to release desired proteins.  There are fewer extracellular proteins from which it is easy to separate product of interest.  Whole cells may be removed from protein containing extracellular media by methods such as centrifugation or filtration.  Few subsequent purification steps are required for final product of recovery. Specific examples of industrially important protein secreted into the extracellular medium during fermentation include•

Various amylolytic and proteolytic enzymes produced by bacilli Cellulases and other activities produced by fungi such as Trichoderma viridiae

Intracellular protein products:

3

In some instances the protein of interest many be intracellular. cell harvesting.

In such cases it

becomes necessary to disrupt the cells upon completion of fermentation and Such approach releases not only the protein of interest but also the enteric intracellular content of the cell. This, in turn, renders more complicated the subsequent purification procedures required to obtain the final product. Specific examples of intracellular proteins of industrial significance include • • • Asperaginase Penicillin acylase and Glucose isomerase

General steps for obtaining a protein of interest: (traditional methods) 1. Traditionally identification of the most suitable microbial protein source involved screening a wide range of candidate microorganisms. 2. The existence of simple, rapid and sensitive assay to identify the protein of interest greatly facilitates such screening activities. 3. Initial screens serve to identify microbial species expressing the protein of interest. 4. Further screens pinpoint microbial species producing the largest quantities of the protein. 5. Frequently, organisms found to produce elevated levels of protein of interest are subjected to mutational studies using chemical mutagens or UV-light, in an effort to isolate overproducing strains. Advantageous mutations can result in product enhancement in two ways:
1. A mutation in the regulation sequence of the gene encoding the desired

protein can result in increased levels of expression of the gene product.
2. A mutational event occurring in the gene itself (direct) can result in


an altered amino acid sequence which may render the protein more functionally efficient or Enhance its stability.

Example:
1. Soil bacteria are amongst the most common group of organisms subjected to

routine screening.

Soil bacilli (apart from B. cereus group) are suitable

enzyme producers as they generally conform to GRAS requirements.
4

They are also easily cultured in simple media and produce variety of industrially important enzymes extracellularly. 2. Hundreds of different species of fungi also inhabit the soil, especially near the soil surface where aerobic conditions prevail. Such fungi are active in degrading a wide variety of biological materials present in the soil. They thrive on such material largely by secreting extracellular enzymes (cellulases, pectinase) capable of degrading large polymeric plant molecules such as cellulose, hemicelluloses and pectin with subsequent assimilation of the liberated nutrients. Protein production in genetically engineered microorganism:  Genetic manipulation by mutation and selection has played a central role in increasing expression levels of a myriad of microbial proteins. This approach, however, could be at best described as haphazard, because, researchers have little control over the genetic alterations achieved, and thus goal (expected improvement in protein productivity) can be achieved only by chance.  On the other hand, recombinant DNA technology can be utilized in a highly directed manner to achieve specific genetic alterations and rational improvements in source productivity.  Strain improvements by ‘trial and error’ mutational methods will continue to play a role, as large numbers of such experiments can be carried out conveniently and relatively inexpensively if suitable screening procedures are in place to detect the desired product, Recombinant DNA technology can be used to increase the levels of production of an endogenous microbial protein by a number of methods. These include: a) Introduction of additional copies of the relevant gene into the microorganism b) Introduction of a copy or copies of the relevant gene into the organism where control of expression has been placed under a more powerful promoter. Such strategies can result in a several-fold increase in production of the protein of interest. We can use genetically engineered microorganism to produce the desired gene because: 1. They are less expensive
5

2. The technique is simple 3. Production is easily controllable 4. Heterologous proteins can be produced 5. Production rate is high.

Heterologous protein expression systems Heterologous protein production in E. coli: The expression of recombinant proteins in cells in which they do not naturally occur is termed heterologous protein production. and prokaryotic origin. Bacterial expression systems are commonly used for production of heterologous gene products of both eukaryotic The expression of heterologous proteins in E.coli, which is A number of the bacterial system, is most widely and routinely used.

therapeutically important proteins are now produced as heterologous in E.coli. The first heterologous protein to be empolyed clinically was human insulin produced in E.coli – first approved 1982, UK, West Germany, Netherland, USA. General considerations of selecting E.coli as heterogeneous protein expression host E.coli is widely used as the host for heterogeneous protein expression for the following advantages: 1. Ease of growth and manipulation using simple laboratory equipments. 2. Availability of dozens of vectors and host strains that have been developed for maximizing expression.
3. A wealth of knowledge about the genetics and physiology of E.coli 4. Expression can often be achieved quite rapidly beginning with a eukaryotic

cDNA clone, express the protein in E.coli and purify in miligram quantities in less than 2 weeks. 5. Suitable fermentation technology well established. 6. Can generate potentially unlimited supplies of recombinant protein. 7. Economically attractive. Limitations using E. coli as heterogeneous protein expression host
6

1. Inability of E. coli as a prokaryotic to carry out post translational modification

which is typical for Eukaryotic. (glycosydation, phosphorylation, acetylation).
2. Limited ability to carryout extensive disulfide bond formation . (assembly of

heterologous proteins are less formed, thus active proteins are not formed).
3. Some proteins are made in insoluble form, a consequence of protein

misfolding, aggregation and intracellular accumulation as inclusion bodies. (when expression level is high-chance of IB is large, also depends on the nature of the proteins; thus form inactive form). 4. Some times sufficient expression may not be observed due to protein degradation or insufficient translation (mRNA may remain in secondary structure and translation hampered)
5. Codon sequence for a specific amino acid in Eukaryotic is different from

Prokaryotic as E.coli. Most common problem: • •

This phenomenon is known as “codon bias” which

vastly hampers protein synthesis and gene expression in E.coli. Inadequate expression levels Poor product solubility

These limitations of gene expression in E.coli can be over come by two major ways: a. Improving the level of expression b. Improving the solubility of the protein Improving the level of expression The level of expression is sometimes inadequate to meet all the need, even when expression systems are used that employ strong transcriptional and translational signals; level of expression can be improved by: a) Induction condition b) Coding sequence of the heterologous gene c) Use of protease deficient host strains
a) Induction condition:

Induction conditions depend on the protein expression system (vector, operon, host media etc). Varying the time and/or temperature of induction of expression of
7

heterologous protein is to find out the optical conditions of product accumulation. Induction can be done with
• •

chemical e.g., IPTG for lac operon; or physical-Pll system allows induction by temperature. Changing the composition of the growth media can also improve expression in some cases.

Some proriens require only the change of temperature, depending on the type of promoters. Some are leaky promoters; thus if the proteins are toxic , cell may die.
b) Coding sequence of the heterologous gene:

Sometimes similar amino acids can interchange, like leucine interchanges with isoleucine. This does not change the configuration of the native protein but increase the level of expression. Expression is often improved by making changes to the nucleotide sequences of the coding region that don’t change the amino acid sequence of the expressed product. Improvement of expression level been reported by changing G and C residues in the first few codons to A and T. As G≡C seems to have more chance of developing secondary structure so their replacement allows more expression as chances of forming secondary structure is translation initiation region decreased. In case of some amino acids several different codons are used; in that case we can use A=T rich codon instead of G≡C rich one. Plasmid that over expressed tRNA molecules that recognized rare codons in the heterlogous gene have also been reported. One can be introduced in to the E. coli strain being used for expression as an alternative to changing rare codons in regular codon sequence. c) Use of protease deficient host strains: The uses of host strains of E.coli carrying mutations which eliminate the production of cellular proteases can sometimes enhance product accumulation by reducing degradation of the protein product. Improving the solubility of the protein

8

The most significant obstacles to the uses of E.coli are protein insolubility under conditions of high level expression. To increase solubility following techniques are proposed: a) Secretion of the heterologous protein b) Growth temperature c) Reduction of rate of protein synthesis d) Co-expression of chaperones and enzymes influencing folding of the heterologous protein in vivo e) In vitro refolding of the heterologous protein
a) Secretion of the heterologous protein:

The periplasmic space comprises ~ 10-40% of the total cell volume under normal growth conditions and contains ~ 4% of total proteins (100pts). An E.coli secretion signals sequence attached with heterologous protein direct it into the periplasm. The signal sequence (18-25 amino acids) is removed during secretion and refolding of the secretory protein into native conformation occurred resulting in accumulation of soluble protein in periplasm. bodies. In some cases, protein may be secreted in the extracellular media. the permeability of the outer membrane. It could be So no misfolding occurs, therefore no inclusion

spontaneous or as a result of genetic or physiological manipulation which increase Release of periplasmic proteins in extracellular space confers further advantages in purification processing. Incases of secretory and no secretory proteins: Such secretion can be achieved by using the host own signal sequence or signal sequence of other E. coli strain or from eukaryotic cells. The efficiency of signal sequence removal is also influenced by the amino acid sequence in heterologus protein, especially first few amino acids following the cleavage site. The protein secretory is native state show better expression after fusion of signal sequence than that of the native non secretory one. Disadvantages:
9

• •

Full mechanisms not understood. Not always effective.
b) Growth temperature:

The improvement of the solubility of proteins expressed in the cytoplasm is achieved by reducing the growth temperature of the culture (to 30 or lower) during incubation. Thus, induction by temperature. Optimizing of solubility in the majer concern than the level if expression. Using weak promoters.
c) Reduction of rate of protein synthesis:

Increased protein synthesis sometimes result in insolubility. production can be reduced by
i)

Thus protein

Use of weaker promoter ( e.g. 05mM ITPG )

ii) Providing partial incubation condition ( not switch on the full-blown ; naïve

induction is suppressed by the inducer)
iii) Less miss-folding and aggregation These lead to accumulation of larger amounts of soluble proteins.

c) Co-expression of chaperones and enzymes influencing folding of the heterologous protein in vivo: Post translational folding of proteins, assembly into oligomers and transport to the periplasm are facilitated bye molecular chaperones. Co expression of proteins with E coli chaperones Gro-Es-Gro-El or DnaJ or DnaK or with Eukaryotic proteins disulfide isomers, has sometimes proven useful. recognized the polypeptide released from association of protein and mis-folding. Molecular chaperones: Post translational folding of proteins, assembly into oligomers and transport to the periplasm are facilitated bye molecular chaperones. They are not enzymes, they and inhibit improper ribosome

10

Molecular chaperons are large group of unrelated protein families to stabilize unfolded proteins or unfold them for translocation across the membrane or for degradation and/or to assist in their correct folding or assembly. • • • • they are proteins but not enzymes, ability to recognize enzymes, partially filded proteins, not related to protein nature or characteristics misfolded proteins , unfold, stabilize, unfolded and transported to the desired location. Properties of molecular chaperones 1. Molecular chaperones interact with unfolded or partially folded.
2. They stabilize non-native conformation and facilitate the correct folding of

protein subunits.
3. Do not interact with non-native protein nor do they form part of final folded

structure. 4. They are mostly non-specific; interact with a wide variety of polypeptide chain. 5. Some are specific and restricted to special target.
6. They often couple with ATP-binding

and carry out hydrolysis of folded

proteins.
7. They are essential for viability, there is often expression incereased by

cellular structures. In vitro refolding of the heterologous protein: Proteins that are made in insoluble form in inclusion bodies can often be solubilized and refolded. It is not native or completely unfolded protein rather partially folded intermediate. This inclusion body can be easily separated by centrifugation to soluble proteins and other cellular components they are less susceptible to degradation. The major disadvantage is that the conditions for refolding into fully active form may be difficult to find. During centrifugation at 500-1000 g inclusion bodies precipitated before cell debris so easily separated. Solubilization is usually accomplished in two ways:

11

1. With denaturants such as urea or guanidine hydrochloride. Removal of high molar concentration of urea (6M) used as denaturant by dialysis or dilution allow to refold 2. By using reducing agents that break disulfide bond. There are some factors that may be varied to improve the yield of active proteins:
1. Protein concentration ( conc

, IB formation

)

2. purity 3. pH 4. ionic strength of the buffer 5. the disulfide oxidizing condition which is adjusted by adding cysteine, arginine, glutathionine or dithiotheitol) The advantages of expression or heterologous proteins as fusion proteins or with protein tag: Many vectors are available which allow expression of heterologous proteins which are fused at their N or C terminal partners are often termed as protein-tag. For example, Histidine (His) tag is a fusion protein. Such fusion partners offer several potential advantages:
A. Improved expression:

Fusion of the N terminals of a heterologous

protein to the C-terminus of a highly expressed fusion partner often allow high level of expression of the fusion protein.
B. Improved solubility: Fusion of N terminus of heterologous protein to the C-

terminus of a soluble fusion partner often improves solubility of a protein.
C. Improved detection: Fusion of a protein at either terminus to a short

peptide or a poly peptide which is recognized by an antibody or binding protein allows western blot analysis of a protein during expression and purification.
D. Improved purification:

It is a widely used phenomenon.

Simple

purification schemes have been described for proteins fused at either end to tags which bind affinity resins. Available tags includes His 6 (six tandem hisitidine residues); which bind to Ni-NTA (Nitrilo-triacetate chelated with Ni2+ ions), GST (Glutathione-S-transferase, which bind to glutathione-sepharose). These tags bind to their specific resins and separated easily. effect of tags on protein and excised easily. Heterologous protein production in yeast
12

There is no

Popular yeast hosts utilized in the production of heterologous proteins of industrial interest include: 1. Saccharomyces cerevisiae 2. Schizosaccharomyces pombe 3. Hansenula polymorpha 4. Kluyveromyces lactis 5. Pichia pastoris 6. Yarrowia lipolytica 7. Candida utilis

Table: 2.6 ( page -66)

Attractive features of yeasts as host for heterologous protein expression: 1. Non pathogenic 2. Rapidly dividing 3. Most yeasts are GRAS listed 4. Easy to grow in laboratory or fermenter 5. Less expertise required 6. Molecular biology/genetic make up well known 7. Eukaryotic cell system so presence of post translational modification is possible 8. Can be used in many biotechnological processes Advantages and disadvantages of heterologous protein production in yeast: Advantages: 1. Most yeast are GRAS listed 2. Proven history of use in many biotechnological process 3. Fermentation technology is well established
13

4. Ability to carry out post translational modifications in recombinant protein Disadvantages: 1. Recombinant proteins usually expressed at very low levels, typically representing only 5% of total cellular protein 2. Retention of many heterologous protein in the periplasmic space 3. Adverse public perception of products manufactured via recombinant technology 4. Some post-translational modifications differ significantly from those achieved by animal cells. Primary and secondary objectives of expression of a heterologous gene in yeast: There are two primary objectives: 1. To achieve as high levels of expression of the recombinant protein as possible 2. To ensure that it is authentic in terms of both its primary amino acids sequences and post-translational modification. Secondary objectives include: 1. Genetic stability of the expression system 2. Cost effectiveness in terms of media and inducers General consideration during application of yeast as a suitable expression system:
1. Approximately 5% genes in S. cerevisiae genome have single intron and S.

cerevesiae is unable to remove that intron from the primary transcript of heterologous gene. Therefore, it is imperative that a cDNA copy of the heterologous coding sequence is used as starting point for any expression.
2. If the target protein requires post-translational modifications like disulfide

bond formation, glycosylation, then the protein need to be targeted to the secretory pathway. Many yeast species, particularly S. cerevisiae have a very

14

low secretory capacity.

Thus, the chances of obtaining a high yielding

secretion system are limited, but it is not impossible. 3. The target heterologous protein may be toxic to the yeast cell, even through the encode protein may not have any associated toxicity in its normal cellular environment. To solve such problem, it is essential to use an expression Such problems arise usually with membrane protein or system which can be tightly regulated. Such tightly regulated system stops leaky expression. membrane associated proteins and they cause inhibition of growth, resulting in change in biomass.
4. There is no guarantee that there will be a successful outcome to a yeast

expression project. be observed.

Such failure may occur in spite of optimizing all the Yeasts are not only

necessary and obvious parameters. Sometimes a low level of expression may The reason behind this is unknown. example of unpredictable pool. So a parallel expression system using other host strains like E. coli is used in simultaneously for the successful expression of the heterologous protein.

Heterologous protein production in Fungi Attractive features of fungi as a host for heterologous protein production: Filamentous fungi represent attractive hosts for heterologous protein production for a number of reasons: 1. They are enzymatically capable of carrying out post-translational

modification. 2. Many are GRAS-listed 3. They have been extensively employed on an industrial scale for many decades in the production of a variety of enzymes as well as other primary and secondary metabolites (e.g., vitamins, organics acids, antibiotics, etc.).
15

4. They are capable of synthesizing and secreting large quantities of certain

proteins into extracellular medium. species of Saccharamyces..

It is a marked contrast to E. coli or

5. Extracellular production of heterologous proteins is desirable as it simplifies subsequent product purification.
6. Expression level is very high.

Some industrial strains of Aspergillus niger

produce even 20 gram/liter levels of protein (glucoamylase). 7. Large scale fermentation systems been developed and optimized over a long time, due to their industrial significance. The major limitation for fungal system in production of heterologous protein is due to the codon usage. Specific codons for amino acids are used differentially in one species compared to the species from which the gene was obtained. Examples of some proteins of industrial significance expressed in

recombinant fungal systems: Protein Human interferon Bovine chymosin Aspertic proteinase Organism Aspergillus niger; A. nidulans A. niger; A. nidulans (from A. oryzae

Rhizomucor michei) Triglyceride lipase Lactoferrin A. oryzae A. niger; A. oryzae

Plant as a source of industrially important protein Plants are traditionally used as a source of different biologically active molecules. Narcotics such as opium are the best known example of such products. species of plants. purified from Crude opium consists of dried milky exudates obtained from unripe capsules of certain The most important medical constituents of opium are different crude opium preparations, generally by ion exchange alkaloids, among which morphine is the renounced one. Morphine is extracted and chromatography.
16

Limitations of higher plants as producers of proteins: For a number of reasons higher plants are not regarded as good procedures of commercially important proteins: 1. Plant growth is seasonal in nature; hence a constant production and supply of product is not possible. 2. Many industrially important proteins synthesized in plants are also found in other biological sources. In most cases, the alternative source becomes the active source of choice for both technical and economic reasons.
3. Higher plants tend to accumulate waste substances in structures called vacuoles. Upon cell disruption these wastes, which include a number of precipitating and denaturing agents, are released, which may irreversibly inactive many plant proteins.

Some plant proteins and their functions: A number of industrially important proteins are obtained from plants; two such proteins include Monellin and Thaumatin:  These are recognized as the sweetest-known naturally occurring substances. They are non nutritive sweeteners. Do not promote tooth decay. Function: Used as sweetening against in food industry and can be safely used as

food ingredients for diabetic patients. Β-amylases  Produced by many higher plants, mostly obtained from barley. These enzymes play an important role in starch-processing industry.

Function: Papain:

Also known as vegetable pepsin. produced on industrial scale. Source: papaya.

It is the best-known plant-derived protein

It is collected from the latex of the green fruit and leaves of Carica

17

Properties:

It is a cysteine protease. Active site contains an essential cysteine It consists of a signal

residue which must remain in reduced state to maintain its proteolytic activity. The purified enzyme exhibits broad proteolytic activity. polypeptide chain containing 212 amino acids, with a molecular mass of 23 kDa. The term papain is applied not only to the purified enzyme but also to the crude dried latex. Mechanism of activity: protein in animals. It is used industrially as a meat tenderizing agent.

Proteolytic activity is directed against the collagen fiber, the major structural Collagen is present in connective tissue and blood vessels which renders meat though. Optimum temperature is relatively high (65oC) and remain active up to 90oC. Due to thermostability, it maintains its proteolytic activity during initial stages of cooking. Application:      Papain has several industrially important applications, such as Properties: 1. It is a cysteine protease, 2. got higher proteolytic activity than papain and has Digestive aid Debriding wounds)  Before slaughter to relax cattle collagens. agent (cleaning of 3. industrial applications. Molecular mass of pure ficin is ~25 kDa. 4. Most large-scale industrial similar

Meat tenderizing Bating of animal skin Clarification of beverages

applications of papain and ficin do not require highly purified enzyme preparations.
5. Plant

enzymes, food

in

particular industry,

Ficin: Another commercially available

those destined for application in the processing must be obtained only from nontoxic, edible plant species. Application: same as papain.
18

protease obtained naturally from plant sources. It is extracted from the latex of certain tropical tree.

source α-amylase Production of heterologous Chymosin are of now plants. Bacillus licheniformi s Calf proteins in plants A number of different heterologous proteins produced and in a peptides variety

n system Tobacco

Tobacco Tobacco

Erythropoiet Human in Glucoamyla se Growth hormone Interferon-β Lysozyme Phytase Human Chicken Aspergillus niger Serum albumin Xylanase Clostridium thermocell um human Aspergillus niger Trout

Potato

Genetic manipulation of plant systems may be undertaken for a number of reasons. Introduction of foreign genes or cDNAs may be performed in order to confer a novel function can or be ability on the and manipulated species. sequences means:
1. Use of Agrobacterium as carrier

Tobacco

Tobacco Tobacco Tobacco

Novel DNA introduced

maintained in plant cells by several

Potato

Tobacco

2. Direct

injection

of

DNA

into

certain plant cells. Using such techniques plants can be engineered to produce insecticides, which when expressed, may play a protective role. Their target is often Sometimes growth regulatory genes. called plant body. Table: Some Recombinant Development of transgenic plant to carry out heterologous gene expression:

A

transgenic

plant

is

usually Ti

antibody produced in transgenic plant

developed using the Ti plasmid of Agrobacterium tumefaciens. plasmid has the ability to infect plant cell and incorporate gene in the plant chromosomal DNA.

proteins of industrial or medical interest and their plant sources: Protein Original Expressio

The

T-DNA segment of the Ti

plasmid is capable of transferring
19

DNA origin.

or

gene

of

heterologous

T-DNA region is bordered

by 25 nucleotides pair imperfect repeats, one of which must be present in cis for T-DNA excision and transfer. The foreign gene n using standard techniques. must be inserted between these two border sequences.

One of the ways of producing transgenic plant is binary vector system which is based on the observation that the T-DNA does not need to be physically attached to the rest of the Ti plasmid. A two plasmid system, with the TDNA on a relatively small molecule, and the rest of the plasmid in normal form, is just as effective as transforming plant cells. The TDNA plasmid is small enough to have unique restriction site and to be manipulated to insert gene of target protei

Fig: - The binary vector strategy. cointegration strategy. (Right figure)

Plasmids A and B complement each other

when present together in the same A. tumifaciens cell.(Left figure) The

Advantages and disadvantages of recombinant protein production in transgenic plants Advantages: 1. Economically attractive production cost
20

2. Ease of scale up 3. Availability of established practices/equipment for plant harvesting/storage. 4. Elimination of downstream processing requirements if the plant material containing the recombinant protein can be used directly as the protein source. 5. Ability to produce target protein in specific plant tissue. 6. New function of protein may evolved 7. Previous functions may enriched 8. Ability to carry out post-translational modification. Disadvantages:
1. Low expression level ( max 4% xylan by plant of total soluble proteins,

sometimes less than 1%). 2. Glycosylation pattern is usually different from that observed on animal glycoprotein. 3. Lack of industrial experience or data on large-scale downstream processing of plant tissue. 4. Seasonal or geographical nature of plant growth 5. Presence of toxic substances is plant cell vacuoles 6. Availability of established, alternative production systems. Heterologous peptide production in plant cell: It is possible to produce a range of commercially important peptides in plant systems like therapeutic proteins. However, plant-based expression systems achieve glycosylation patterns that differ (in extent and composition) to those achieved by animal cells. This point is important if an altered glycosylation pattern in any way negatively influences the recombinant protein product. This is especially important in the context of therapeutically important glycoproteins, where an altered glycosylation pattern could influence product safety and/or efficacy. Certain oligosaccharide epitopes commonly found on plant glycoproteins
21

are highly immunogenic in mammals. potentially be more immunogenic.

This suggests that some mammalian

glycoproteins intended for therapeutic application, if expressed in plant cells, could

Example of small therapeutic peptides produced in plants: Thyrotrophin: It is a 3 amino acids long peptide, naturally produced in It stimulates the synthesis and release of hormone

hypothalamus of mammals. from interior pituitary gland.

Some other peptides are produced in minute amount because of difficulties in synthesis and purification; currently such peptides are chemically synthesized but the cost of peptide is very high in market. Cost of chemical synthesis increase with the length of peptide, e.g., 3 amino acids peptide cost less than 5 amino acids one. Sometimes chemical modifications of peptides are done after observing the sequence. Now-a-days, some other peptides are produced biotechnologically using E. coli and yeast expression system. Seeds of higher plants for protein accumulation:  The ability to target expression of recombinant proteins to a specific plant tissue can be advantageous. It could reduce the potential toxicity of the protein (for the plant) and reduce environmental and regulatory concern.  Targeted accumulation of the protein in plant seed is particularly attractive. The seeds of higher plant naturally contain high levels of storage protein (~50% of the seed protein). Seeds can be stored for extended period after harvest, inexpensively and without causing protein degradation. In contrast, plant green tissue deteriorates rapidly after harvest and requires immediate protein extraction after harvest, or storage or harvest under refrigeration or frozen condition (expensive). Such seed proteins are utilized to synthesize smaller peptides in higher plant. Leuenkephalin is an early example of transgenic peptide production in plant. The recombination was done by inserting the DNA coding sequence of Leuenkephain into the gene coding for a seed storage protein termed 25 albumin. The family of 2S albumins are among the smallest seed storage proteins known, having a molecular mass in the order of 12 kDa. This family of proteins is derived from a
22

group of structurally related genes - all of which exhibit both conserved and variable sequences. The strategy employed to produce leu-enkephalin involved the following steps: 1. Substitution of part of the variable region sequence of 2S albumin with DNA sequence coding for 5 amino acids neurohormone. This DNA construct is flanked on both sides with codons coding for tryptic cleavage sites. 2. Expression of the altered 2S albumin gene resulted in production of a hybrid storage protein containing the leu-enkephalin sequence. 3. The enkephalin is subsequently released from the altered protein by tryptic cleavage and purified by HPLC 4. Due to the incorporation of tryptic cleavage sites, the purified product contained an extra lysine residue which is subsequently removed by treatment with carboxypeptidase C. (a proteolytic enzyme used to remove amino acids residue from C-terminal end) Though it is not a chemically & technically feasible method, it is economically good, since a number of larger polypeptide have also been expressed in the seeds of various plants.

Animal tissue as a protein source  A wide variety of commercially available proteins are obtained from animal sources, especially numerous therapeutic proteins such as insulin and blood factors. The existence of slaughterhouse greatly facilitates the collection of significant quantities of the particular tissue required as protein source.  The best known protein obtained from animal source is insulin. It is a

polypeptide hormone that is produced in the pancreas by the beta cells of the islets of Langerhans. The hormone is used therapeutically in the treatment of insulin-dependent diabetes (Type 1 diabetes, diabetes mellitus). slaughterhouse cattle and pigs. for approximately 10 days.
23

Until the early

1980’s insulin was obtained exclusively from pancreatic tissue derived from The amount of insulin obtained from the pancreatic tissue of three pigs satisfies the requirement of one diabetic patient

The increasing worldwide incidence of diabetes raised the demand for insulin which exceeded supply from slaughter house sources. produced by recombinant means. This is no longer of concern however as potentially unlimited supplies of insulin can now be

Table: Some proteins of industrial and medical significance traditionally obtained from animal sources. Protein Insulin Source Porcine/bovine pancreatic tissue Porcine/bovine pancreatic tissue Application Treatment of type 1 diabetes Reversal of insulin induced hypoglycaemia Induction of Porcine pituitary glands Follicle-stimulatory hormone (FSH) Urine of postmenopausal women superovulation in animals Treatment of (human) reproductive dysfunction Treatment of reproductive dysfunction Treatment of anaemia Treatment of haemophilia Various diagnostic and therapeutic applications Cheese manufacture

Glucagon

Human chronic gonadotrophin Erythropoietin Blood factors

Urine of pregnant women Urine Human plasma

Polyclonal antibodies Chymosin (rennin)

Human or animal blood Stomach of calves

Significant

drawbacks

of

using

proteins

from

animal

sources

therapeutically and its remedy:  Most industrially significant proteins obtained from human and other animal sources are destined for therapeutic use. One most significant disadvantage
24

with regard to the therapeutic utilization of such proteins from animal sources

(especially from human) relates to the potential presence of pathogens in the raw material.  The large numbers of haemophiliacs who contracted acquired immune deficiency syndrome (AIDS) from HIV-infected blood transfusion stands as testament to this fact. Outbreaks of bovine spongiform encephalitis (BSE/Mad cow diseases) in cattle herds from various countries serve as another example. A number of precautions should be taken when animal tissue is used as a protein source. These include: 1. The use of tissue obtained from disease-free animal only 2. Downstream processing procedure of protein purification must be validated, thereby must eliminate pathogens which may be present in starting material.  Many pathogens, in particular viruses are markedly species specific. better not to administer to other animals of that same species. Heterologous protein production in transgenic animals- Objectives and limitations:  Over the past number of years great advances have been recorded in the field of transgenic animals. The preliminary objective was to improve various animal characteristics, such as improving the animal growth rate dramatically by genomic integration of extra copies of growth hormone gene. direct microinjection of DNA into ova, although success rate is low.  Molecular farming: All these production of transgenic animal, called molecular farming. Done by twodesirable characteristics: One goal of such method is the introduction of specific functional genes into animals, thereby conferring on them desirable characteristics such as more efficient feed utilization, improved growth characteristics or generation of leaner meat. Increase the target gene product: Another goal of such transgenic technology is to confer on the transgenic animal the ability to produce large quantities of industrially important proteins like therapeutic proteins.
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Thus,

therapeutically used proteins obtained from a particular animal species are

It is done by

The production of industrially important proteins has been achieved with varying degrees of success in mice, goats, sheep and cattle. Initial successes in expressing high levels of growth hormone in transgenic animals highlighted some problems associated with the technique: 1. Chronically high circulatory levels of growth hormone (significantly beyond normal level) resulted in many adverse physiological effects. 2. Elevated circulatory levels of many proteins of potential therapeutic value would also almost certainly promote similar adverse effects on normal transgenic animal metabolism.

Specific animal tissues were also targeted as heterologous protein expression system; examples include: Mammary gland was the first choice of tissue targeted as heterologous protein expression site. By targeting expression of the foreign gene into the mammary gland, the heterologous protein may be secreted directly into the milk.

Signal sequence promoter of a milk protein + Foreign gene construction Gene encoding the heterologous protein

Egg fertilization

Micro injection into an egg of target animal

Implementation of fertilized egg into a surrogate mother That transgenic animal is now capable of secreting the desired protein selectively in its milk.

Embryo is then brought to term

Foreign DNA has been successively incorporated in to the newborn’s chromosomal DNA and expressed.

In mid 1980s, a therapeutic protein, human tissue plasminogen activator (t-PA) was expressed in mammary gland of transgenic animal (mice). This was
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achieved by injecting a DNA construct consisting of promoter and upstream regulatory sequence from the mouse whey acidic protein gene to the gene coding for human t-PA into mice embryo. Whey acidic protein is the most Biologically active t-PA was t-PA is a serine abundant whey protein found in mice milk.

recovered from the milk of the resultant transgenic mice. used therapeutically in thrombosis (heart vein block). mammalian cell line.

protease that converts plasminogen to plasmin. It dissolves fibrin clots and is It is also produced in

Desirable features of mammary gland as heterologous expression system: ( over cell culture0 Mammary glands have a number of potential and desirable properties as heterologous protein expression system than other alternative production methods. As such1. High production capacities:

During a typical 5-month period, one sheep If the recombinant protein is

can produce 2-3 litters of milk per day. product/week.
2. Ease of collection of source material:

expressed at a level of 1g/l, a single sheep could produce in excess of 20g

This only requires the animal to

be milked.

Commercial automated milking systems are already available;

such systems require only moderate design alteration as they are already designed to maximize hygienic standards during the milking process.
3. Low capital investment requirements and low operational costs:

Traditional

production

methods

yielding

recombinant

proteins

require

considerable expenditure on fermentation equipment. Using this technology, such costs are reduced to raising and maintaining the transgenic herbs.
4.

Ease of production scale up: expanded by breeding programmes.

Producer animal numbers can be

Draw backs of mammary glands: 1. A number of technical details are yet to be optimized. 2. Yield of heterologous proteins are extremely variable. than 1mg/l has been found.
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In some cases, less

3. Mammary gland tissue is capable of carrying out a broad range of posttranslational modifications of the protein synthesized. Detailed characterization of the nature of such modifications, particularly in relation to glycosylation patterns remain to be carried out. Heterologous protein production using animal cell culture Animal cell culture also represents an important source of several medically important proteins, virtually all of which are destined for therapeutic or diagnostic application; monoclonal antibodies, various vaccines, interferons, factor VIII, t-PA etc are some of the best known example: Table :- Some recombinant pharmaceutical proteins approved for general medical use that are produced commercially via animal cell culture: Protein Factor VIII Factor IX t-PA FSH Erythropoietin Interferon-β Produced in CHO cells; BHK Cells CHO cells CHO cells CHO cells CHO cells CHO cells Medical application Hemophilia A Hemophilia B Heart attacks Infertility Anemia Multiple sclerosis Various, including Several monoclonal antibodies prevention of kidney Hybridoma cells transplant rejection and localization of tumors in vivo

Difference between animal and microbial cell that influence animal cell culture and fermentations design: Animal and microbial cells exhibit many basic differences in their cellular physiology and structure. Some basic differences that influence animal cell culture and fermentation design are mentioned below: 1. Animal cells do not possess a cell wall, and thus are more susceptible to physical damage when compared to their microbial counterpart.
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2. The nutritional requirements of animal cells are more complex then those of microbial cells
3. Animal cells tend to have lower oxygen requirements, so used CO2 incubator.

4. The animal cells grow more slowly in artificial media then their microbial counterpart. 5. Far greater numbers of animal cells are required to seed the fermentation tank effectively. Fermentation tanks in which animal cells are cultured usually contain agitation blades of modified design in order to reduce the potential physical damage caused by shear forces generated by such rotating blades. Moreover, fermentations are usually conducted in air-lift reactors, in which liquid culture motion is promoted within the vessel by sparging an air-CO2 mixture into the reactor in order to further reduce shear force.

Fig: - Design of a generalized microbial cell fermentation vessel (a), and an animal cell bioreactor (b). Animal cell bioreactors display several structural differences as compared to microbial fermentation vessels, in particular, (i) the use of a marine type impeller (some animal cell bioreactors-air-lift fermenters are devoid of impellers, and use sparging of air-gas as the only means of media agitation); (ii) the absence of baffles and; (iii) curved internal surfaces at the bioreactor base. These modifications aim to minimize damage to the fragile animal cells during culture.

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Serum replacements are mixture of nutrient supplemented with variety of purified proteins like insulin, epidermal growth factor, transferrin, prolactin, prostaglandin E1, ethanol amine, phosphoethanol amine Hydrocortisone etc.

Bacterial and fungal contamination is one of the fundamental problems of animal cell culture. contamination. To overcome this problem high concentration of antibiotics are used to avoid microbial like penicillin, streptomycin, gentamycin etc.

Prerequisite:        Sterile tissue culture flask Pipettes (sterile 1 ml, 5 ml, 10 ml) Sterile Pasteur pipette Sterile plastic conical tubes Sterile cryo-tubes Hemocytometer  Laminar flow  Maintain personal hygiene Sterile hood

CO2 incubator Liquid N2 freezer Inverted microscope Vacuum line Double distill H2O used in

  

sterilization

Media compositions and other prerequisites for animal cell culture: Animal cells are more complex compared with microbial cells and their cultures are more fragile compared with bacterial, fungal and yeast cultures due to their requirement of complex media and their nutritional requirements which are more expensive than microbial culture media. Previously several living system fluids like serum, lymph, embryo, homogenate etc are used as major source of nutrients. But their exact composition was unknown. Currently two different types of growth media for animal cell culture are available. Those are –
a) Typical serum containing growth medium - that contains:

 

Serum (5-20%) Inorganic salt

 

Nitrogen sources Carbon and energy source
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  

Vitamins Trace elements Trace elements

 

Growth factors Buffers in water

b) Serum free growth medium - that contains:

1. Amino acids 2. Serum replacements 3.Vitamins 4.Major inorganic salts 5.Other organics 6.Trace elements 7.Buffers and indicators

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Generation of primary cell line: Most of the primary cell line taken from embryo of different animals as they dissociate easily and have a longer life time. and have limited/shorter life span. 1. Tissues animals. 2. Mechanical/biochemical treatments to dissociate them 3. Frozen in liquid N2 freezer. 4. Specific amounts of cells taken out. excised from specific 5. Primary cell line 6. Subculture on medium, maintain time interval 7. Repeating subculture 8. Maintain cell line 9. Transferring culturing) 10.Secondary cell line Trypsin is usually used to treat cell culture to dissolve extracellular attachment and to get single cells in floating conditions. Cells are washed with PBS with no calcium (Ca2+) because Ca2+ play role in cell attachments. Maintenance of cell culture: A number of defined media have been developed to grow and maintain cell line. Among them 1. Eagle’s modified Eagle’s medium (MEM) 2. Dulbecco’s modified Eagle’s medium (DMEM) are most popular Madam used DMEM media-composition given below: 1. 10 ml glutamine (2%) 2. 5 ml penicillin + streptomycin (1%) 3. 50 ml serum (10%) Tissue culture flask containing exponentially growing cell line: 4. 450 ml media 5. Total 500 ml (some media lost during sterilization) old media (subAdult cells may not dissociate easily

organs of animals or embryo of

1. Observe the presence of cell line under an inverted microscope. 2. Aspirate the old media with the aid of a Pasteur pipette. 3. Add 3-5 ml PBS slowly 4. Aspirate PBS

5. Add 3-5 trypsin (30 sec room temperature) 6. Aspirate trypsin
7. Everything must be pre-warmed

(37oC) before use

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