LOVELY

PROFESSIONAL
UNIVERSITY

TERM PAPER OF CONCEPTS IN

BIOTECHNOLOGY ON PRODUCTION OF
RECOMBINANT PROTEIN IN EUKARYOTES

SUBMITTED TO,
HARSH KUMAR.

SUBMITTED BY,
R109B44

ACKNOWLEDGEMENT

I here by acknowledge that this term paper “PRODUCTION OF RECOMBINANT

PROTEINS IN EUKARYOTES” has been made under the guidance of Mr. Harsh
Kumar. Yet there is always room for improvement, I invite you for the improvement of
this term paper.
CONTENTS
1. INTRODUCTON
2. ADVANCES IN THE PRODUCTION OF HUMAN THEREUPEUTIC
PROTEINS IN YEAST AND FILAMENTOUS FUNGI
3. RECOMBINANT PROTEIN PRODUCTION IN YEAST
- EXPRESSION VECTORS
 - FACTORS AFFECTING THE LEVEL OF RECOMBINANT PROTEIN
PRODUCTION
4. PRODUCTION OF RECOMBINANT PROTEINS IN OTHER
MICROPRGANISM
5. PRODUCTION OF HUMAN FACTOR V|||
6. ADVANTAGES OF USING ANIMAL CELL LINES FOR RECOMBINANT
PROTEIN PRODUCTION
7. OVER PRODUCTION OF RECOMBINANT PROTEIN
- USE OF STRONG CONSTITUTIVE PROMOTER
- INCLUSION OF AN INTRON
- INCLUSION OF POLYDENYLATION SITE
- REMOVAL OF UNNECESSARY AND UNTRANSLATED SEQUENCES
- OPTIMIZATION OF TRANSGENE SEQUENCE FOR EFFICIENT
TRANSLATION
- INCORPORATION OF TARGETTING SIGNAL
8. RESEARCH ON RECOMBINANT PROTEIN
9. REFERENCE

INTRODUCTION

Recombinant DNA Technology involves the isolation and cloning of genes of interest,
production of the necessary gene construts using appropriate enzymes and then transfer
and expression of these genes into an appropriate host organism. This approach is also
called genetic engineering. This technique has been used to achieve the following two
broad objectives:
(1) Production of recombinant proteins
(2) Modification of the organism’s metabolic pattern for the production of new,
modified or more quantity of metabolities.
In this term paper I am going to discuss about recombinant protein.

RECOMBINANT PROTEINS

Proteins produced by genes transferred by genetic engineering into selected host cells are
called recombinant proteins since their production is based on recombinant DNA
technology. Recombinant proteins form an important component of biopharmaceuticals,
i.e. , biotechnology products having pharmaceutical applications. A large number of
recombinant proteins are being produced in mammalian cell cultures, some of which,
viz., human growth hormone, tissue plasminogen activator, erythropoietin and blood
clotting factor V|||, are already in therapeutic use. The host cells used for large scale
production of the various recombinant proteins is as follows: Chinese hamster ovary line,
baby hamster kidney line BHK, mouse mammary tumors line C127, mouse my eloma
cell lines and mammalian cell lines.
The use of cell line has been made possible by the following
developments in cell culture technology:
1 Development of culture systems permitting large scale culture of animal cells at
high densities
2 Development of media, which minimize or even obviate the use of serum.
These developments have enhanced the yields and reduced the costs of same products
obtained from genetically engineered bacteria and yeast.
Even when an animal cell line produces a desired protein on its own, expression
vectors and cloned genes are still used to maximize yields by using a much stronger
promoter than the one present with the endogenous gene. Often the promoter used in
expression vectors comes from a virus like SV40. The cost of production from
mammalian cell lines increases remarkably due to the strict implementation of
rigorous quality control measures to ensure that the product is free from viruses that
infect mammals. Insect cell cultures provide an attractive alternative host system for
recombinant protein production. The expression vector is based on baculoviruses,
which do not attack vertebrates. The product of polyhedron gene of baculoviruses
accumulates in the insect cell nuclei as large inclusion bodies towards the end of
infection cycle; polyhedron makeup over 50% of the total cell protein. The transgene
is inserted in the place of polyhedron gene, but all the signal sequences of the
transcription unit are retained. The recombinant protein is produced at the same level
as polyhedron . Several mammalian proteins have been produced in insect cell
cultures, but unfortunately the proteins are not glycosylated correctly; this remains the
chief limitation of this system.
Advances in the production of human therapeutic
proteins in yeasts and filamentous fungi
Yeast and fungal protein expression systems are used for the production of
many industrially relevant enzymes, and are widely used by the research
community to produce proteins that cannot be actively expressed in Escherichia
coli or require glycosylation for proper folding and biological activity. However,
for the production of therapeutic glycoproteins intended for use in humans,
yeasts have been less useful because of their inability to modify proteins with
human glycosylation structures. Yeast glycosylation is of the high-mannose type,
which confers a short in vivo half-life to the protein and may render it less
efficacious or even immunogenic. Several ways of humanizing yeast-derived
glycoproteins have been tried, including enzymatically modifying proteins in
vitro and modulating host glycosylation pathways in vivo. Recent advances in the
glycoengineering of yeasts and the expression of therapeutic glycoproteins in
humanized yeasts have shown significant promise, and are challenging the
current dominance of therapeutic protein production based on mammalian cell
culture.

RECOMBINANT PROTEIN PRODUCTION IN YEAST

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

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

Factors Affecting the Level of Recombinant Protein Production.

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

PRODUCTION OF RECOMBINT PROTEIN IN OTHER

MICRO-ORGANISM

(1) Pichia pastoris is another species of yeast; it produces recombinant proteins up to

30% of the total cell proteins. Its glycosylation abilities are very similar to those
of animal cells, although the sugar structures do show trivial differences from
those in animal proteins. Expression vectors use the AOX promoter from the gene
 that encodes alcohol oxidase; this promoter is induced by methanol. But P.
pastoris sometimes degrades recombinant proteins; this can, however, be
controlled by using special growth media.
(2) Filamentous fungus Aspergillus nidulans has good glycosylation properties and
secretes proteins into the growth medium. Expression vectors for A. nidulans
usually use glucoamylase promoter, which is induced by starch.
(3) Trichoderma reesei has the same desirable features as A. nidulans. Expression
vector for T. reesei use the cellobiohydrolase promoter, which is induced by
cellulose.
(4) Other yeasts that have been used for recombinant protein production include
Hansenula polymorpha, Yarrowia lipolytica and Kluveromyces lactis, the last of
these can be grown on wastes from the food industry.

PRODUCTION OF HUMAN FACTOR V|||

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

Advantages of using Animal Cell Lines for Recombinant Protein

production

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

Overproduction of Recombinant proteins

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

The various strategies for maximizing transgene expression are briefly described in the
following sections.
Use of Strong Constitutive Promoter

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

Inclusion of an Intron

The presence of an intron in a eukaryotic expression unit usually enhances transgene

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

Inclusion of a Polydenylation site

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

Removal of Unnecessary and Untranslated Sequences

(1) Eukaryotic mRNA contain untranslated regions of variable le

Some other 5’-UTRs codons contain sequences that affect mRNA stability, e.g., AU-rich
sequences reduce Mrna stability, and such sequences have been discovered in some 5’-
UTRs, In addition , the 5’-and 3’-UTRs may be rich in secondary structure, which
prevent efficient translation. In view of the above, the 5’-and 3’-UTR sequences are
generally removed from transgene construts to maximize their expression and the
production of recombinant at their 5’-as well as 3’-ends. These UTRs can influence the
level of gene expression in a number of different ways as follows.
(2) Some 5’-UTRs contain AUG codons upstream of the authentic translation
initiation codon; often such additional AUG codons interfere with translation
initiation. Proteins.

Optimization of Transgene Sequence for Efficient Translation

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

Incorporation of Targetting Signal.

Often eukaryotic proteins are modified after translation to become functional. For
example, many proteins of therapeutic value require authentic glycosylation patterns
for their correct function as well as for preventing an immune responses in the
patients, Specific types of post-translational modifications occur in particular cellular
compartments.Therefore, it is necessary to target the recombinant proteins to that
specific compartment of the cell where the given modification will be effected. For
example, the proteins to that specific compartment of the cell where the given
modification will be effected. For example, the proteins that are to be glycosylated,
must be targeted to the secretary pathways using a signal peptide. Many mammalian
expression vectors contain sequences that encode specific signal peptide sequences.
For example, the Invitrogen vectorpSec.Tag2 contains a signal sequence from the
murine immunoglobin light-chain signal peptide; the signal peptide encoded by ttisb
sequence targets the protein to the secretary pathway with a high efficiency. In
addition, the C-terminus of the recombinant protein obtained by using this vector is a
fusion of two different epitope tags, which facilitate protein purification.
Several high level expression systems are available. When a large scale
production system is envisaged, the choice of a particular system will depend on the
cost of culture system, need for stable or transient transfection, and the possible need
for inducible expression. For therapeutic applications, a stable, permanently-
expression cell line consistently yields products of reproducible quality. In case of
transient expression, the scale of transfection step becomes important. It may be
pointed out that the maximum expression attainable may not always be required.
Inducible gene expression can be achieved by using metallothionein promoter. In this
case, transcription is induced by metal ions like cadmium and zinc. Alternatively, a
promoter achieved by steroid/thyroid hormone/retinoic acid may be used. For
example, mouse mammary tumour virus (MMTV) LTRhas a complex promoter
region that is activated by steroid hormone.
RESEARCH ON RECOMBINANT PROTEIN
The Article Cloning and Characterization of Multgenesi Encoding the Immunodominant
30-Kilodalton Major Outer Membrane Proteins of Ehrlichia canis and Application of the
Recombinant Protein for Serodiagnosis Send in September 1998 to theDepartment of
Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University,
Columbus, Ohio 43210-1093 It was Received at 2 March 1998/Returned for modification
7 April 1998/Accepted 16 June 1998

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

REFERENCE

1. Essentials of Biotechnology-By B.D. Singh

2. Biotechnology By-R.C. Dubey
3. From Web Sites-
www.wikipedia.com

www.google.com