EUKARYOTIC EXPRESSION SYSTEMS AND THEIR

APPLICATIONS

Presented by-:Vimlesh Gupta

L-2011-V-59-MDepartment of Veterinary

Microbiology…

INTRODUCTION

Expression system-: Overproduction of proteins by placing the

gene encoding them under the control of a strong promoter.

Goal:require for those proteins that are produced at low level , for structural & functional studies and for medical & industrial purpose.

THESE TECHNIQUES RELY ON:

the universality of the Genetic Code

knowing the Genetic Code the relative similarity of the

translational machinery (ribosome). Rapid progress in molecular

biology/genetic engineering cloning/amplification . DNA sequencing. Cutting and pasting pieces of DNA

from one source into another.• The Expression vector

EXPRESSION VECTORS

• It is engineered plasmid to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.

• Goal -is the production of large amounts of stable messenger RNA, and therefore proteins.

Origin of replication:  DNA polymerase selectable marker(s):  antibiotic

resistance promoter:  recognized by RNA

polymerase multiple cloning sites (restriction

enzyme sites):  cutting/pasting of DNA fragments.

Termination site: to stop the transcription.

EXPRESSION OF TARGET GENE…..

Once the expression vector inside the cell, expression of target genes occurs.

3’ Poly A Tail

Protein Coding Region

3’ UTR 5’ UTR

G

5’ Cap

Exon 2 Exon 3Exon 1

Int. 2

Int. 1

AAAAA

PROTEIN EXPRESSION SYSTEMS

CHOICE OF THE EXPRESSION SYSTEM

7

Cell-free Bacteria Yeast Insect Mammalian

Easy of use

Cost of media and Equipment

Pos-translational Modifications(Probability of protein function)

Time Requirement

EXPRESSION IN E. COLI

E. coli remains the host cell of choice for the majority of protein expression experiments.

Its rapid doubling time (approximately 30 min) in simple defined(inexpensive)media.

Extensive knowledge of its promoter and terminator sequences.

Of course, E. coli does suffer from the fact that is a prokaryotic organism when it is used to produce eukaryotic proteins.

1) Problems resulting from the sequence of the foreign gene(a)Introns are not removed in E. coli(b) Premature termination of

transcription(c) a problem with codon bias.

2)PROBLEMS CAUSED BY E. COLI Unfortunately, the proteins of

bacteria and higher organisms are not processed identically.

E.g. Glycosylation of recombinant proteins synthesized in E. coli are never glycosylated correctly.

E. coli might not fold the recombinant protein correctly, so usually it is insoluble and forms an inclusion body within the bacterium.

E. coli might degrade the recombinant protein.

EXPRESSION IN EUKARYOTIC SYSTEM

The problems associated with obtaining high yields of active recombinant proteins from genes cloned in E. coli have led to the development of expression systems for other organisms.

Yeast and filamentous fungi. Insects. Higher eukaryotic cells.

Four promoters frequently used in expression vectors for microbial eukaryotes. P = promoter.

EXPRESSION IN YEAST

As eukaryotes, yeasts have many of the advantages of higher-eukaryotic cells, such as post-translational modifications.

Yeast cell growth is faster, easier and less expensive than other eukaryotic cells, and generally gives higher expression levels.

Three main species of yeast are used for the production of recombinant proteins – Saccharomyces cerevisiae, Pichia pastoris and Schizosaccharomyces pombe.

SACCHAROMYCES CEREVISIAE

Baker’s yeast, S. cerevisiae, is a single-celled eukaryote that grows rapidly (a doubling time of approximately 90 min) in simple, defined media similar to those used for E. coli cell growth.

Proteins produced in S. cerevisiae contain many, but not all, of the post-translation modifications found in higher eukaryotic cells.

Saccharomyces cerevisiae is currently the most popular microbial eukaryote for recombinant protein production.

A number of strong constitutive promoters have been used to drive target gene expression in yeast.

For example, the promoters for the genes encoding phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GPD) and alcohol dehydrogenase (ADH1) have all been used to produce target protein.

The GAL promoter is induced by galactose,regulating expression of a cloned foreign gene.

PROMOTERS FOR S. CEREVISIAE EXPRESSION VECTORS

1) THE GAL SYSTEM

In yeast, like almost all other cells, galactose is converted to glucose-6-phosphate by the enzymes of the Leloir pathway.

Each of the Leloir pathway structural genes (collectively called the GAL genes) are expressed at a high level, but only when the cells are grown on galactose as the sole carbon source.

Each of the GAL genes contains within its promoter at least one, and often multiple, binding sites for the transcriptional activator Gal4p.

The binding of Gal4p to these sites, and its transcriptional activity when bound, is regulated by the source of carbon available to the cell.

When yeast is grown on glucose-: transcription from the GAL4 promoter is down-

regulated Reduced production of Gal4p activator in the cell, reduced level of activator binding at the promoters of

the GAL structural genes.Carbon sources, such as raffinose-: Gal4p is produced and binds to the GAL structure gene

promoters, but a repressor, Gal80p, inhibits its activity. Gal80p binds directly to Gal4p and is thought to mask

its activation domain such that it is unable to recruit the transcriptional machinery to the gene.

Only in the presence of galactose-: is the inhibitory effect of Gal80p alleviated, leading to strong, inducible levels of target gene expression.

To produce a target protein in S. cerevisiae using galactose induction, the gene encoding the protein must be cloned so that it is under the control of a GAL promoter.

The promoter from the GAL1 gene, encoding galactokinase, is most commonly used, but synthetic promoters containing multiple Gal4p binding sites are also available.

Once constructed, the expression vector is transformed into yeast cells and protein production is initiated by switching the cells into a galactose-containing medium. Proteins produced in this way seldom accumulate to the levels of recombinant protein found in E. coli cells.

A difficulty is brought about as a consequence of the activator of the GAL genes, Gal4p, being normally present in the yeast cell at a very low level.

Therefore, if the expression vector, which carries multiple Gal4p binding sites, is a high-copy-number plasmid then there may be insufficient Gal4p to activate the expression of all of the available target genes to a maximum level.

To overcome this problem-: GAL4, is also placed under the control of PGAL1

Figure: Galactose inducible gene expression in yeast. The expression of genes from multicopy vectors under the control of the GAL1 promoter (PGAL1) can be increased substantially if the gene encoding the transcriptional activator of GAL1, GAL4, is also placed under the control of PGAL1. In this case, induction by galactose will produce more Gal4p and consequently more of the target protein.

2) THE CUP1 SYSTEM

Cells must maintain a proper cellular level of copper ions (Cu2+ and Cu+) that is not too low to cause deficiency and not too high to cause toxicity.

In S. cerevisiae, copper homeostasis consists of uptake, distribution and detoxification mechanisms.

At high concentrations, copper ion detoxification is mediated by a copper ion sensing metalloregulatory transcription factor called Ace1p.

Upon interaction with copper, Ace1p binds DNA upstream of the CUP1 gene, which encodes a metallothionein protein, and induces its transcription.

The transcription of CUP1 is induced rapidly by addition of exogenous copper to the medium.

Expression vectors harbouring the CUP1 promoter can therefore be used to induce target gene expression in a copper-dependent fashion.

Advantage-: Unlike the GAL system, yeast cultures containing

the CUP1 expression plasmid can be grown on rich carbon sources, such as glucose, to high cell density, and protein production is initiated by the addition of copper sulphate to the cultures.

Disadvantage-: One potential drawback with this system is the

presence of copper ions in yeast growth media, and indeed in water supplies.

Therefore, the ‘off’ state in the absence of added copper may still yield significant levels of protein production Yields of recombinant protein are relatively high.

Recombinant Proteins Successfully Produced in S. cerevisiae

•For a range of reasons as expressed previously each of these represented a better product than was obtainable using a prokaryotic expression system

WHY OTHER YEAST SPECIES?

S. cerevisiae sometimes hyperglycosylates proteins-Proteins also sometimes retained in periplasmic

space S. cerevisiae also produces ethanol at high

cell densities which is toxic to cells.

PichiaPastoris Highly efficient promoters available like-: AOX1 (alcohol oxidase for methanol metabolism)

promoter easily turned on by methanol

The promoter regulating the production of alcohol oxidase (AOX1) can be used to drive heterologous protein expression in P. pastoris since it is tightly regulated and induced by methanol to very high levels.

The first step in the metabolism of methanol is the oxidation of methanol to formaldehyde using molecular oxygen (O2) by the enzyme alcohol oxidase.

Alcohol oxidase has a poor affinity for O2, and P. pastoris compensates for this deficiency by generating large amounts of the enzymes.

P. PASTORIS INTEGRATING EXPRESSION VECTOR

•Integrating system requires double recombination (AOX1p and 3’AOX1 regions)•His selection in a HIS4- strain•Usual prokaryotic/eukaryotic sequences•Alcohol oxidase expression system (AOX1)

Advantages-: High level of target protein- For example, the expression of

the gene encoding recombinant hepatitis B surface antigen results in the production of more than 1 g of the antigen from 1 L of P. pastoris cells. This is much greater than could be achieved in S. cerevisiae.

Additionally, in comparison to S. cerevisiae, P. pastoris may have an advantage in the glycosylation of secreted proteins.

Glycoproteins generated in P. pastoris more closely resemble the glycoprotein structure of those found in higher eukaryotes.

Low level of ethanol. Disadvantages-: The only significant problem with P. pastoris is that it

sometimes degrades recombinant proteins before they can be purified.

SCHIZOSACCHAROMYCES POMBE

S. pombe is a single-cell eukaryotic organism with many properties similar to those found in higher-eukaryotic organisms.

Additionally, eukaryotic proteins expressed in S. pombe are more likely to be folded properly, which may reduce protein insolubility associated with the production of many proteins in E. coli.

Protein production in S. pombe is usually controlled by the expression from the nmt1 (no message in thiamine) promoter.

This promoter is active when the cells are grown in the absence of thiamine, allowing downstream transcription of genes under its control, while in the presence of greater than 0.5 μM thiamine, the promoter is turned off.

Overall protein production levels are similar to those found in S. cerevisiae.

EXPRESSION IN INSECT CELLS

The expression system is based on the baculoviruses, a group of viruses that are common in insects but do not normally infect vertebrates.

The baculovirus genome includes the polyhedrin gene, whose product accumulates in the insect cell as large nuclear inclusion bodies toward the end of the infection cycle .

Similar levels of protein production also occur if the normal gene is replaced by a foreign one.

Baculovirus Systems

Baculoviruses are rod-shaped viruses that infect insects and insect cell lines.

They have double-stranded circular DNA genomes in the range of 90–180 kbp.

Viral infection results in cell lysis, usually 3–5 d after the initial infection, and the subsequent death of the infected insect.

The nuclear polyhedrosis viruses are a class of baculoviruses that produce occlusion bodies in the nucleus of infect cells.

These occlusion bodies consist primarily of a single protein, polyhedrin, which surrounds the viral particles and protects them from harsh environments.

CONT..

•Polyhedron gene is not essential for life cycle (protects virus in environment)•Commonly used with cultured insect eggs

BACULOVIRUS TRANSFER VECTOR

•Done in cell culture

The production of a recombinant baculoviral

genome for the productionof proteins in insect cells. The

target gene is cloned under the control of the polyhedrin

promoter into a transfer vector that also contains regions of the viral

genome that flank thepolyhedrin locus. The vector is then co-transfected into insect cells with

a viral genome thathas been linearized using

restriction enzymes (RE) that cut in several places. Homologous

recombination between the linear genome and the vector will result

in formation of afunctional viral genome that is

capable of producing viral particles. The inclusion of lacZ

in the transfer vector allows for visual screening of viral plaques to identify recombinants. recombinant

virus look different because they lack the coat protein.

.

Baculovirus Expression System

.usually contain the lacZ gene, or another readily observable reporter gene, which allows for the visual identification of recombinant plaques by their blue appearance after staining with X-Gal.

ADVANTAGES The polyhedrin gene is not required for the continuous

production of infectious virus in insect cell culture. Its sequence is replaced with that of the heterologous gene.

The polyhedrin gene promoter is very strong. This determines a very high level of production of recombinant protein.

This system is capable of post-translational modifications.

Disadvantages• Expensive. • Glycosylation in insect cells is different. • Discontinuous expression: baculovirus infection of insect cells kills the

host and hence the need to reinfect fresh cultures for each round of protein synthesis.

• Inefficient for production on a commercial scale

EXPRESSION IN HIGHER-EUKARYOTIC CELLS

For the production of mammalian proteins, mammalian cells have an obvious advantage.

In most cases these proteins have been processed correctly and are indistinguishable from the non-recombinant versions.

• Two modes of expression - transient and stable.

• Three cell types are dominant in transient expression: human embryonic kidney (HEK), COS and baby hamster kidney (BHK), whilst CHO (Chinese hamster ovary) cells are used predominantly for stable expression.

MAMMALIAN EXPRESSION VECTORS

Eukaryotic origin of replication -is from an animal virus: e.g. Simian virus 40 (SV40).

Selective marker-antibiotic resistance genes.

Promoter sequences that drive expression of both marker and cloned heterologous gene.

The transcription termination Polyadenilation signals-are usually from

animal viruses (human CMV, SV40, herpes simplex virus) or mammalian genes (bovine growth hormone, thymidine kinase).

MAMMALIAN EXPRESSION VECTOR

•“I” is an intron that enhances expression •Other signals similar to insect and prokaryotic vectors

TWO VECTOR EXPRESSION SYSTEM

•Useful for proteins of two different polypeptides

TWO GENE EXPRESSION VECTOR

SELECTIVE MARKER GENE SYSTEMS FOR MAMMALIAN CELLS

These systems, however suffer from leaky gene expression in the absence of induction and potentially damaging induction conditions.

To overcome some of the problems of using endogenous promoters to drive target gene expression, systems have been imported from bacteria to control gene expression in mammalian cells.

TET-ON/TET-OFF SYSTEM

The E. coli tet operon was originally identified as a transposon (Tn10) that confers resistance to the antibiotic tetracyclin.

The TetR protein, in a similar fashion to the lac repressor protein (LacI), binds to the operator of the tetracycline-resistance operon and prevents RNA polymerase from initiating transcription.

Regulator plasmid – produces a version of the E. coli tetracycline repressor (TetR) that is fused to the transcriptional activation domain of the herpes simplex virus VP16 protein. The fusion protein is constitutively produced in the host cell from the CMV promoter.

Response plasmid – contains the target gene cloned downstream of multimerised copies of the tetracycline operator (tetO) DNA sequence that form a tetracycline response element (TRE) cloned into a minimal CMV promoter that is not, on its own, able to support gene activation.

Figure :Tetracycline regulated gene expression for protein production in mammalian cells. The Tet-off and Tet-on systems differ in their transcriptional response to added tetracycline. The Tet-off system turns transcription of the target gene off in response to tetracycline, whereas the Tet-on system, which contains a mutant version of TetR with altered DNA binding properties, activates gene expression in response to tetracycline addition

In the absence of tetracycline, the TetR-VP16 fusion protein will bind to the TRE and activate transcription of the target gene.

Upon the addition of tetracycline to the cells, however, TetR will dissociate and target gene transcription will be turned off.

That is, the addition of tetracycline turns target gene expression off.

The use of the tet system has become more prevalent due to the existence of a mutant version of TetR.

Tet-off uses the wild-type TetR protein fused to VP16. Target gene expression is active in the absence of tetracycline but not in its presence.

Tet-on uses the mutant rTetR proteins fused to VP16. Target gene expression is active in the presence of tetracycline but not in its absence.

Advantages: The advantage of this on and off switching system is that host

cells do not need to be exposed for long times to the antibiotic prior to the induction of either gene expression or gene silencing.

There are no examples of higher eukaryotic proteins, which could not be made in detectable levels, and in a form identical to the natural host (that includes all types of post-translational modifications). 

Disadvantages: Cultures characterised by lower cell densities and lower

growth rates. Maintenance and growing very expensive. Gene manipulations are very difficult. Mammalian cells might contain oncogenes or viral DNA, so

recombinant protein products must be tested more extensively

PHARMING—RECOMBINANT PROTEIN FROM LIVE ANIMALS AND

PLANTS

The use of silkworms for recombinant protein production is an example of the process Often referred to as pharming, where a transgenic organism acts as the host for protein synthesis.

Pharming is a recent and controversial innovation in gene cloning.

APPLICATIONS OF RECOMBINANT PROTEINS

Eukaryotic expression systems are frequently employed for the production of recombinant proteins for structural & functional studies and for medical & industrial applications like-:

Hormones Insulin:  Diabetes Human thyroid stimulating hormone

Blood clotting factors Coagulation factor VIII :  hemophilia A. Coagulation factor IX:  hemophilia B.

CONT….

Interferons interferon-(alpha)-2a:  chronic hepatitis C. gamma interferon:  hepatitis B, C, herpes and

viral enteritis. Immunization agents

Hepatitis B vaccine:  a non-infectious vaccine derived from Hepatitis B surface antigen (HBSA) produced in yeast cells.

Research enzymes Restriction endonucleases Endoglycosidases:  PNGase

Recombinant Proteins Successfully Produced in S. cerevisiae

•For a range of reasons as expressed previously each of these represented a better product than was obtainable using a prokaryotic expression system

EXAMPLES OF PROTEINS SUCCESSFULLY PRODUCED BY BACULOVIRUS SYSTEMS

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