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-a-
days, 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 • Aspergillus
• Bacillus amyloliquefaciens • Penicillium
• Various other bacilli • Mucor and
• Lactobacilli and • Rhizopus
• Streptomyces spp.
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. In such cases it
becomes necessary to disrupt the cells upon completion of fermentation and
cell harvesting.
 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. Bacterial expression systems are
commonly used for production of heterologous gene products of both eukaryotic
and prokaryotic origin. The expression of heterologous proteins in E.coli, which is
the bacterial system, is most widely and routinely used. A number of
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. This phenomenon is known as “codon bias” which
vastly hampers protein synthesis and gene expression in E.coli.
Most common problem:
• 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. So no misfolding occurs, therefore no inclusion
bodies.

In some cases, protein may be secreted in the extracellular media. It could be

spontaneous or as a result of genetic or physiological manipulation which increase
the permeability of the outer membrane. 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. Thus protein

production can be reduced by

i) 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. They are not enzymes, they
recognized the polypeptide released from ribosome and inhibit improper
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.

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. There is no
effect of tags on protein and excised easily.

Heterologous protein production in yeast

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Popular yeast hosts utilized in the production of heterologous proteins of industrial
interest include:

1. Saccharomyces cerevisiae 5. Pichia pastoris

2. Schizosaccharomyces pombe 6. Yarrowia lipolytica

3. Hansenula polymorpha 7. Candida utilis

4. Kluyveromyces lactis

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
system which can be tightly regulated. Such tightly regulated system stops
leaky expression. Such problems arise usually with membrane protein or
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. Such failure may occur in spite of optimizing all the
necessary and obvious parameters. Sometimes a low level of expression may
be observed. The reason behind this is unknown. Yeasts are not only
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. It is a marked contrast to E. coli or
species of Saccharamyces..

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 Organism

Human interferon Aspergillus niger; A. nidulans

Bovine chymosin A. niger; A. nidulans

Aspertic proteinase (from A. oryzae

Rhizomucor michei)

Triglyceride lipase A. oryzae

Lactoferrin 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. Crude
opium consists of dried milky exudates obtained from unripe capsules of certain
species of plants. The most important medical constituents of opium are different
alkaloids, among which morphine is the renounced one. Morphine is extracted and
purified from crude opium preparations, generally by ion exchange
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.

Function: These enzymes play an important role in starch-processing industry.

Papain:

Also known as vegetable pepsin. It is the best-known plant-derived protein

produced on industrial scale.

Source: It is collected from the latex of the green fruit and leaves of Carica
papaya.

17
Properties: It is a cysteine protease. Active site contains an essential cysteine
residue which must remain in reduced state to maintain its proteolytic activity. The
purified enzyme exhibits broad proteolytic activity. It consists of a signal
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: It is used industrially as a meat tenderizing agent.

Proteolytic activity is directed against the collagen fiber, the major structural
protein in animals. 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

 Meat tenderizing Properties:

 Bating of animal skin 1. It is a cysteine protease,

 Clarification of beverages 2. got higher proteolytic activity

than papain and has similar
 Digestive aid industrial applications.

 Debriding agent (cleaning of 3. Molecular mass of pure ficin is

wounds) ~25 kDa.

 Before slaughter to relax cattle 4. Most large-scale industrial

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

5. Plant enzymes, in particular

Ficin: those destined for application in
the food processing industry,
Another commercially available
must be obtained only from non-
protease obtained naturally from plant
toxic, edible plant species.
sources. It is extracted from the latex
of certain tropical tree. Application: same as papain.
18
 source n system

α-amylase Bacillus Tobacco

licheniformi
Production of heterologous s
proteins in plants
Chymosin Calf Tobacco
A number of different heterologous Erythropoiet Human Tobacco
proteins and peptides are now in
produced in a variety of plants. Glucoamyla Aspergillus Potato
Genetic manipulation of plant systems se niger
may be undertaken for a number of
Growth Trout Tobacco
reasons.
hormone
Introduction of foreign genes or cDNAs Interferon-β Human Tobacco
may be performed in order to confer a
Lysozyme Chicken Tobacco
novel function or ability on the
Phytase Aspergillus Tobacco
manipulated species. Novel DNA
niger
sequences can be introduced and
Serum human Potato
maintained in plant cells by several
albumin
means:
Xylanase Clostridium Tobacco
1. Use of Agrobacterium as carrier
thermocell

2. Direct injection of DNA into um

certain plant cells.

Using such techniques plants can be Development of transgenic plant

engineered to produce insecticides, to carry out heterologous gene
which when expressed, may play a expression:
protective role. Their target is often
 A transgenic plant is usually
growth regulatory genes. Sometimes
developed using the Ti plasmid of
antibody produced in transgenic plant
Agrobacterium tumefaciens. Ti
called plant body.
plasmid has the ability to infect
Table: - Some Recombinant plant cell and incorporate gene in
proteins of industrial or medical the plant chromosomal DNA.
interest and their plant sources:
 The T-DNA segment of the Ti
Protein Original Expressio plasmid is capable of transferring

19
 DNA or gene of heterologous
origin. 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
must be inserted between these
n using standard techniques.
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 T-
DNA on a relatively small molecule,
and the rest of the plasmid in
normal form, is just as effective as
transforming plant cells. The T-
DNA plasmid is small enough to
have unique restriction site and to
be manipulated to insert gene of
target protei

Fig: - The binary vector strategy. Plasmids A and B complement each other
when present together in the same A. tumifaciens cell.(Left figure) The
cointegration strategy. (Right figure)

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. This suggests that some mammalian
glycoproteins intended for therapeutic application, if expressed in plant cells, could
potentially be more immunogenic.

Example of small therapeutic peptides produced in plants:

Thyrotrophin: It is a 3 amino acids long peptide, naturally produced in

hypothalamus of mammals. It stimulates the synthesis and release of hormone
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. Leu-
enkephalin is an early example of transgenic peptide production in plant.

The recombination was done by inserting the DNA coding sequence of Leu-
enkephain 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). Until the early
1980’s insulin was obtained exclusively from pancreatic tissue derived from
slaughterhouse cattle and pigs. The amount of insulin obtained from the
pancreatic tissue of three pigs satisfies the requirement of one diabetic patient
for approximately 10 days.

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

Table: Some proteins of industrial and medical significance traditionally

obtained from animal sources.

Protein Source Application

Porcine/bovine Treatment of type 1

Insulin
pancreatic tissue diabetes

Porcine/bovine Reversal of insulin

Glucagon
pancreatic tissue induced hypoglycaemia

Induction of
Porcine pituitary glands superovulation in
Follicle-stimulatory animals
hormone (FSH) Treatment of (human)
Urine of post-
reproductive
menopausal women
dysfunction

Treatment of
Human chronic Urine of pregnant
reproductive
gonadotrophin women
dysfunction

Erythropoietin Urine Treatment of anaemia

Treatment of
Blood factors Human plasma
haemophilia

Various diagnostic and

Polyclonal antibodies Human or animal blood
therapeutic applications

Chymosin (rennin) Stomach of calves Cheese manufacture

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
with regard to the therapeutic utilization of such proteins from animal sources
24
 (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. Thus,

therapeutically used proteins obtained from a particular animal species are
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. It is done by
direct microinjection of DNA into ova, although success rate is low.

 Molecular farming: All these production of transgenic animal, called molecular

farming. Done by two-

desirable 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.

25
 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

Micro injection into an egg

Egg fertilization
of target animal

Implementation of fertilized Embryo is then brought to

egg into a surrogate mother term

That transgenic animal is now capable of Foreign DNA has been

secreting the desired protein selectively in its successively incorporated in to
milk. 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

26
 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
abundant whey protein found in mice milk. Biologically active t-PA was
recovered from the milk of the resultant transgenic mice. t-PA is a serine
protease that converts plasminogen to plasmin. It dissolves fibrin clots and is
used therapeutically in thrombosis (heart vein block). It is also produced in
mammalian cell line.

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 such-

1. High production capacities: During a typical 5-month period, one sheep

can produce 2-3 litters of milk per day. If the recombinant protein is
expressed at a level of 1g/l, a single sheep could produce in excess of 20g
product/week.

2. Ease of collection of source material: 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: Producer animal numbers can be

expanded by breeding programmes.

Draw backs of mammary glands:

1. A number of technical details are yet to be optimized.

2. Yield of heterologous proteins are extremely variable. In some cases, less

than 1mg/l has been found.

27
 3. Mammary gland tissue is capable of carrying out a broad range of post-
translational 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 Produced in Medical application

Factor VIII CHO cells; BHK Cells Hemophilia A

Factor IX CHO cells Hemophilia B

t-PA CHO cells Heart attacks

FSH CHO cells Infertility

Erythropoietin CHO cells Anemia

Interferon-β CHO cells Multiple sclerosis

Various, including
prevention of kidney
Several monoclonal
Hybridoma cells transplant rejection and
antibodies
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.
28
 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.

29
 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. To overcome this problem high concentration of antibiotics
like penicillin, streptomycin, gentamycin etc. are used to avoid microbial
contamination.

Prerequisite:

 Sterile tissue culture flask  CO2 incubator

 Pipettes (sterile 1 ml, 5 ml, 10 ml)  Liquid N2 freezer

 Sterile Pasteur pipette  Inverted microscope

 Sterile plastic conical tubes  Vacuum line

 Sterile cryo-tubes  Double distill H2O used in

sterilization
 Hemocytometer
 Sterile hood
 Laminar flow
 Maintain personal hygiene

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%)  Nitrogen sources

 Inorganic salt  Carbon and energy source

30
  Vitamins  Growth factors

 Trace elements  Buffers in water

 Trace elements

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

31
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. Adult cells may not dissociate easily
and have limited/shorter life span.

1. Tissues excised from specific 5. Primary cell line

organs of animals or embryo of
6. Subculture on medium, maintain
animals.
time interval
2. Mechanical/biochemical
7. Repeating subculture
treatments to dissociate them
8. Maintain cell line
3. Frozen in liquid N2 freezer.
9. Transferring old media (sub-
4. Specific amounts of cells taken
culturing)
out.
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%) 4. 450 ml media

2. 5 ml penicillin + streptomycin 5. Total 500 ml (some media lost

(1%) during sterilization)

3. 50 ml serum (10%)

Tissue culture flask containing exponentially growing cell line:

1. Observe the presence of cell line 5. Add 3-5 trypsin (30 sec room
under an inverted microscope. temperature)

2. Aspirate the old media with the 6. Aspirate trypsin

aid of a Pasteur pipette.
7. Everything must be pre-warmed
3. Add 3-5 ml PBS slowly (37oC) before use

4. Aspirate PBS