Hetero Logo Us Gene Expression

8/9/2019 Hetero Logo Us Gene Expression

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HETEROLOGOUS

GENEEXPRESSION

IN

EUKARYOTICCELLS

PRESENTED BY :-

ASH. K


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HETEROLOGOUS PROTEIN PRODUCTION IN

EUKARYOTIC CELLS

When recombinant DNA techniques are used to express certain genes outside their

natural expression habitat, the process is described as HETEROLOGOUS GENE

EXPRESSION.

NEED & APPLICATION :-

Recombinant protein expression is the foundation of todays biomolecular research and

thriving Biotech industry.GOAL:- Overproduction of proteins for

# Proteins which are produced by other methods are generally crude preparations & usuallymixture of various other components . In contrast there are number of commercial

applications where highly purified proteins are needed Analytical enzymes or Abs are used

in genetic engineering technology and therapeutic proteins in the treatment of diseases etc.

# Genetic Engineering produces proteins that offer advantages over proteins isolated from

other biological sources. These advantages include :-

High purity High specific activity

Steady supply

Batch to batch consistency

AVAILABLE EXPRESSION SYSTEMS :-

1. Prokaryotic - like E.coli

2. Eukaryotic - like yeast, insect, mammalian cell culture etc

Prokaryotic systems are generally easier to handle and are satisfactory for most purposes.

However there are serious limitations in using prokaryotic cells for the production ofeukaryotic proteins.

LIMITATIONS OF PROKARYOTIC EXPRESSION SYSTEM :-

Many of the Euk proteins undergo a variety ofpost translational modificationslike proper folding , glycosylation, phosphorylation , formation of disulfide bridges.

Endotoxin contamination

STRUCTURAL

STUDIES

ENZYMATIC

STUDIES

COMMERCIAL/

PHARMACEUTICAL

APPLICATIONS

ANTIGENPRODUCTION


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Low yield of functional protein

Difficult purification ( Inclusion bodies)

To avoid these problems investigators have developed eukaryotic expression systems for theproduction of proteins that can be used as therapeutic agents in either humans or animals.

# In general eukaryotic expression vectors have the same kinds of features as theirprokaryotic counterparts :-

A Selectable eukaryotic marker gene

A eukaryotic promoter sequence

The appropriate eukaryotic transcriptional & translational stop signals.

A sequence that signals polyadenylation of transcript mRNA

If the vector is to be used in plasmid form ( i.e as extrachromosomal replicating DNA)then it must also have an ORI that functions in the host cell. Alternatively if the

vector is designed for integration into the host chromosomal DNA , then it must carry

a sequence i.e is complementary to a segment of host chromosomal DNA ( the

chromosomal integration site) to facilitate recombination.

# Because many recombinant DNA procedures are technically more difficult with eukaryotic

cells than with prokaryotic cells , most eukaryotic vectors are designed to be shuttle vectors.In other words , they carry 2 types of origins of replication & selectable marker genes , one

type that functions in E.coli & another that functions in the eukaryotic host cell.

GENERALIZED EUKARYOTIC EXPRESSION VECTOR

The major features of a eukaryotic expression vectors are :-

A eukaryotic transcription unit with a promoter (P)

A cloning site (cs) for the cloned gene

A DNA segment with termination & polyadenylation signals (t)

A eukaryotic selectable marker (ESM) gene system

An origin of replication that functions in the euk cells ( ori-euk) An origin of replication that functions in the E.coli ( ori- E )

An E.coli selectable marker gene Ampr


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Saccharomyces cerevisiae EXPRESSION SYSTEMS:-

For a variety of reasons , the common yeast Saccharomyces cerevisiae has been used

extensively as a host cell for expression of cloned eukaryotic genes :- It is single celled, well known genetically & physiologically & it can be grown

readily in both small culture vessels and large scale bioreactors.

Several strong promoters have been isolated from yeast and characterized & a naturally

occurring plasmid called 2m plasmid can be used in endogenous yeast expressionvector systems.

It is capable ofcarrying out many post translational modifications.

Only 0.5% native proteins are secreted so isolation of secreted proteins is simplified.

It has been listed by the U.S Food & Drug Administration as a Generally Recognised

as Safe (GRAS).

Recombinant proteins produced by S. cerevisiae :-

1. Hepatitis B Surface Ag 1. Hepatitis C virus protein 1. Epidermal G.F

2. Malaria circumsporozite- 2. HIV- 1 Ag 2. Insulin like G.F

Protein 3. Proinsulin3. HIV- envelope protein 4. 1 antitrypsin

5. Blood coagulationFactor XII a

Saccharomyces cerevisiae VECTORS :-

Different classes like:-

1. Episomal orPlasmid Vectors ( YEp)

2. Yeast Replicating plasmids ( YRp)

3. Yeast Centromere plasmids (YCp)

4. Yeast artificial chromosome ( YAC ) vectors

DIAGNOSTICS HUMAN THERAPEUTIC AGENTSVACCINES


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DIRECT EXPRESSION IN S.cerevisiae

The term direct expression is often used to describe vector systems producing

proteins that accumulate in the cytoplasm of a host cell .

The production of the human enzyme Superoxide dismutase illustrates the process of

heterologous gene expression in S. cerevisiae . SO anion is a byproduct of oxygen utilization in aerobic organisms . In humans this

anion helps both to stimulate the inflammatory response of phagocytes and to direct

leukocytes to the site of infection. However excess of this molecule & its derivativescan cause cellular damage. To minimize these potentially cytotoxic effects the

naturally occurring cytoplasmic enzyme Cu/Zn Superoxide dismutase (Cu/Zn- SOD)

scavenges the SO radical & combines with the hydrogen ion to form H2O2 which in

turn is degraded by catalase or peroxidase.

SO anion is also produced when blood is allowed to reenter an organ (reperfusion)

after it has been deprived of blood during a surgical procedure. To prevent this source

of SO anion damage , investigators have proposed that Cu/Zn SOD be administered

to an organ as it is being reperfused. In addition Cu/Zn SOD has the potential to act as a therapeutic agent against

inflammatory diseases such as osteoarthritis , rheumatoid arthritis , scleroderma &ankylosing spondylitis.

For the use in either of these ways an AUTHENTIC FORM of Cu/Zn SOD is

preferred , to avoid any adverse immunological responses that might result from using

an enzyme from another species.

Initially a cDNA for the enzyme was cloned into an E.coli expression system. But due

to certain limitations , the researchers cloned the human Cu/Zn SOD cDNA into ayeast episomal vector in an effort to obtain an authentic enzyme. ( Yeast cells are not

particularly effective at removing introns , so cDNA sequences or chemically

synthesized genes must be used for encoding specific gene products).

S.cerevisiae Expression vector

In human Cu/Zn SOD cDNA yeast vector contained 1. A yeast gene for Leucine Biosynthesis ( LEU2 )

2. A 2m plasmid DNA sequence , which provides a yeast ORI & permits this

plasmid to replicate in yeast cells.


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3. Both an E.coli selectable marker gene (Ampr) & an ORI that functions in E.coli &

allows the routine genetic manipulation involved in the construction of the plasmid to be

done conveniently in E.coli.4. The human Cu/Zn SOD c DNA cloned between the promoter region of the

yeast glyceraldehydes phosphate dehydrogenase gene (GAPDp) & a sequence containing

the signals for transcription termination & polyadenylation of mRNA from the same gene( GAPDt)

# A leucine deficient yeast strain (LEU2 -) was transformed with this vector & the cells were

plated onto medium that lacked leucine .Only cells with the functional LEU2 gene, whichwas supplied by the vector could grow under these conditions. The GAPD promoter is not

regulated ; rather it is tanscribed continuously during the cell growth. Thus human Cu/Zn

SOD cDNA is synthesized throughout the growth period (constitutively).In this experiment ,the yeast cells produced high levels of intracellular Cu/Zn SOD which like authentic protein

from human cells, was acetylated on the amino group of amino terminal alanine residue.

SECRETION OF HETEROLOGOUS PROTEINS BY S.cerevisiae

In yeast , only secreted proteins are glycosylated , so a secretion system must be used

for heterologous proteins that require either O- linked or N- linked sugars for biologicactivity.

To facilitate protein secretion in yeast , the prepro-- factor or leader peptide(signal peptide) coding sequence of the mating type factor 1 gene, is cloned infront(just upstream) of the cDNA that encodes the desired protein. This gene fusion creates a

protein that can efficiently secreted by yeast. During the exporting process , disulfide bond formation , proteolytic cleavage & posttranslational modifications occur & in many cases an active protein is eventually released

to extra cellular environment.

The leader peptide enables a protein to pass through the cytoplasmic membrane & be

secreted. During this process , the leader peptide is removed by a yeast endoprotease thatrecognizes the dipeptide Lys-Arg.

The Lys-Arg codons are therefore placed immediately upstream from the cDNAsequence so that , following removal of leader peptide , the target protein will have the

correct amino acid residue at its N terminus .

A properly processed & active form of the protein hirudin was synthesized & secreted

by S. cerevisiae strain containing an episomal expression vector that included the prepro- - factor.

OTHER YEAST EXPRESSION SYSTEMS


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LIMITATIONS withS.cerevisiae :-

1. PLASMID LOSS During scale up plasmid loss is a frequent occurrence , even when

inducible promoters are used .

2. HYPERGLYCOSYLATION Heterologous protein is often hyperglycosylated ,

containing more than 100 mannose residues in each N linked oligosaccharide side

chain ( instead of natural 8-13 residues ) . The extra mannose units may alter productsbiological activity or change its immunogenicity .

3. PURIFICATION DIFFICULTIES In a number of trials , proteins that weredesigned to be secreted within the periplasmic space , a situation that makes

purification more difficult .

Therefore other yeast species & eukaryotic systems that can act as host cells for the production of heterologous proteins are being examined. Candidate yeasts that are being

considered as alternatives to S. cerevisiae are-

Kluyveromyces lactis has been used commercially for the production of lactase

Schizosaccharomyces pombe- yeast that reproduces by fission rather than by budding.

Yarrowia lipolytica which uses alkanes as a growth substrate.

Pichia pastoris & Hansenula polymorpha which can utilize methanol as the sole

source of carbon & energy.

ADVANTAGES ofPichia pastoris :-

1. It has a highly efficient & tightly regulated promoter of the methanol inducible

gene that encodes alcohol oxidase ( AOX ) the first enzyme of methanol utilization

pathway. In the presence of methanol , as much as 30% of the cellular protein is

alcohol oxidase. In its absence , the AO 1 gene is completely turned off. The AOX1gene promoter responds rapidly to the addition of methanol to the medium. Therefore

the AOX1 promoter is an excellent candidate for both driving the transcription of

cloned genes & producing large amounts of recombinant protein.

2. Because P. pastoris does not synthesize ethanol , very high cell densities are

attained with the secretion of large quantities of protein.

3. P. pastoris normally secretes very few proteins thus simplifying the purification ofsecreted recombinant proteins.

EXPRESSION OF HEPATITIS B VIRUS SURFACE ANTIGEN


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Double recombination Yeast (integration in Pichia pastoris)

AOX1 gene (


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CULTURED INSECT CELL EXPRESSION SYSTEMS

Baculoviruses exclusively infect invertebrates, including many insect species. During the

infection cycle 2 forms of baculovirus are produced

# Many of these packages are released into the environment after cell lysis and death of a

single host organism. Polyhedron protects the virions from being inactivated byenvironmental agents. Upon ingestion by a host, the polyhedron protein is solublized & the

virions are capable of establishing an infection cycle. During late stages of the infectioncycle, the poyhedrin protein is synthesized in massive quantities. That synthesis is initiated

about 36-48 hrs after infection & continues 4-5 day , until the infected cell lyse & host

organism dies .

# Promoter for polyhedron gene (polyh) is a very strong promoter but viral reproduction

cycle doesnot depend on the presence of polyhedrin gene.

# Therefore polyhedron gene was replaced with the gene for a heterologous protein, followed

by infection of cultured insect cells with the genetically engineered baculovirus that resultedin the production of large amounts of heterologous protein, because of similarity of posttranslational modification systems between insects & mammals would mimic closely if not

precisely the authentic form of target protein. So baculoviruses were developed as an

expression vector for both mammalian proteins and animal virus proteins.

# The specific baculovirus used extensively as an expression vector is Autographacalifornica multiple nuclear polyhedrosis virus ( AcMNPV) .

One form consists of single

virions released by an infected

host cell & are capable of

infecting other host cells

Second form is made up of a number of

virions trapped (occluded) in a protein

matrix. Protein of the matrix= Polyhedrin

Package ofoccluded virions = Polyhedron


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# Autographa californica( the alfalfa looper ) & over 30 other insect species like Mamestra

brassicae & Estigmene acrea can be infected with AcMNPV.

# This virus also grows well on many insect cell lines . The most commonly used cell line for

genetically engineered AcMNPV is derived from the fall armyworm (Spodoptera frugiperda)

BACULOVIRUS TRANSFER VECTOR

The transfer vector is an E.coli based plasmid that carries a segment of DNA from AcMNPVconsisting of

the polyhedron promoter region & an adjacent portion of upstream AcMNPVDNA , which provides a region for homologous recombination with AcMNPV .

A cloning site for the input DNA

Polyhedrin termination & polyadenylation signal region & provides an adjacentportion of downstream AcMNPV DNA , which provides a second region for homologousrecombination with AcMNPV .The coding region for the polyhedrin gene has been

deleted from this block of DNA.

A gene of interest is cloned between the polyhedron promoter & termination

sequences & the construct is propagated in E.coli.

Next cotransfection is used to introduce both the transfer vector DNA carrying a

cloned gene & the intact wild type AcMNPV DNA into host cells. ( Recall that intransfection the baculovirus is introduced into cells as isolated DNA. During the normal

viral infection process, baculovirus DNA is introduced biologically by the intact virus ).

Within some of the cotransfected cells , a double crossover event(fig) occurs and the

cloned gene with polyhedron promoter & termination regions then becomes integratedinto the AcMNPV DNA , with the concomitant loss of the polyhedron gene.

Virions lacking the polyhedron gene produce distinctive zones of cell lysis ( occlusionnegative plaques ) from which recombinant baculovirus can be isolated.


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When host cells are infected by recombinant baculovirus , product protein can be

harvested after 4-5 days.

The visual identification of occlusion negative plaques is tedious and subjective.Consequently DNA hybridization or PCR assay can be used to detect recombinant

baculovirus.

The baculovirus expression vector system has been used to produce more than 500

different heterologous proteins like -interferon , -interferon , erythropoietin, HIV -1

envelope protein , interleukin-2 , mouse monoclonal antibodies, rabies glycoprotein,

tissue plasminogen activator, adenosine deaminase, poliovirus proteins etc.

Replacement of polyhedron

gene of AcMNPV with anexpression unit from a

transfer vector- A double

crossover event results in

integration of the expression

unit into AcMNPV genome


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ADVANTAGES & DISADVANTAGES OF INSECT CELL EXPRESSION SYSTEM

ADVANTAGES DISADVANTAGES

1. High level of protein expression

yields upto 100mg of protein 109 cells

1. Glycosylation not as complex as in

human system.2. This system is capable of post

translational modifications.

2. Viral proteases degrade target protein.

3. Capacity of large DNA inserts

accommodate genes upto 15kb.

3.Grow very slowly ( 10-12 days for

setup)

4. Stable integration (due to virus) 4. Cell culture is sustainable for 4-5 days.

5. Easy purification cell lyse themselves

after 96 hrs ( due to virus).

5. Setup is time consuming, not as simple

as yeast.

6. Simultaneous expression of multiple

genes with multiple promoter transfer

vectors.

6. Discontinous expression- Baculovirus

infection of insect cells kills the host &

hence the need to reinfect fresh cultures

for each round of protein synthesis.


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MAMMALIAN CELL EXPRESSION SYSTEM

Sometimes required for difficult- to express proteins or for completeAUTHENCITIY ( matching glycosylation & sequence).

2 modes of expression Transient & Stable .

A number of established cell lines have been developed. Cells from African green

monkey (COS) , Baby hamster kidney (BHK), Human embryonic kidney ( HEK- 239)are used for short term (transient) gene expression foe either rapid production of smallamounts of heterologous proteins or testing the integrity of constructs during various

stages of vector development. CHO cells are commonly used for long term (stable) gene

expression & when high yields of protein are required.

GENERALIZED MAMMALIAN EXPRESSION VECTOR :-

P = Eukaryotic Promoter ( frequently from either human virus CMV , SV 40, HSV etc ormammalian genes- -actin, thymidine kinase, bovine GH ). Promoter sequence drives

expression of both marker & cloned heterologous gene .

I = Intron ( enhances the production of heterologous protein )MCS = Multiple cloning site

pa= Polyadenylation sequence

TT = Transcription termination sequence

SMG = Selectable Marker gene

Amp-r = Ampicillin gene for selecting transformed E .coli

ori euk= Eukaryotic ORI generally from animal virus Simian virus 40 (SV40)

oriE = from E.coli ( for propagation)

Inducible promoters are often used when continuous synthesis of heterologousprotein is toxic to the host cell

Expression of gene of interest (GOI) is increased by placing the sequence for an intron

b/w the promoter & the MCS of the transcription construct ( cassette)

The sequence that are required for selection & propagation of a mammalian

expression vector in E.coli are derived from a standard E.coli cloning vector such as

pBR322.

For the best results , a GOI must be equipped with translational control sequences.


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Initiation of translation in higher eukaryotic organisms depends on a specific sequence

of nucleotides surrounding the start (AUG) codon called the KOZAK SEQUENCE i.e

CC (A or G ) CCAUGG.

K = Kozak sequence , S = Signal sequence , T = Protein affinity tag , P = Proteoloyticcleavage site , SC = Stop codon , UTR = Untranslated sequence ( increases efficiency of

translation to contribute to mRNA stability )

Kozak sequence followed by signal sequence to facilitate secretion, a protein tag

to enhance purification of heterologous protein, a proteolytic cleavage sequence enables

the tag to be removed from heterologous protein. A stop codon is added to ensure thattranslation ceases at the correct location.

Majority of mammalian cell expression vectors carry a single GOI that encodes afunctional polypeptide . However many commercially important proteins consist of 2

different protein chains. For eg human thyroid stimulatory hormone is a 2 chain protein

( heterodimer ) & both Haemoglobin and antibodies are tetramers with 2 copies of each

subunit 22 & H2L2 respectively.

It is possible to clone the gene or cDNA for each subunit of a multimeric protein ,synthesize & purify each subunit separately & then mix the chains together in a test tube.

Unfortunately very few multichain proteins are properly assembled invitro. By contrast ,invivo assembly of dimeric & tetrameric proteins is quite efficient.

Consequently VARIOUS STRATEGIES have been devised f or the production of 2

different recombinant proteins within the same cell .

1. TWO VECTOR EXPRESSION SYSTEM

2. TWO GENE EXPRESSION SYSTEM

3. BICISTRONIC EXPRESSION VECTOR


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LIMITATIONS :-

1. Loss of 1 of the 2

vectors in doubly

transfected cells iscommon.

2. Moreover the 2

vectors are not always

maintained with the

same copy number, so

one subunit is

overproduced relative

to other & yields of the

final product are

reduced.


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MAMMALIAN CELL EXPRESSION

ADVANTAGES DISADVANTAGES

1. Almost human glycosylation &

phosphorylation pattern

1. Production rates may be comparable

low

2. Highest functionality 2. Selection of single clones is very timeconsuming.

3. Lowest immunogenicity & very high

compatibility to humans.

3. Some animal cell lines require a solid

surface on which to grow , adding

complications to the design of culture

vessel. .

4.High safety profile , easy permission as

a drug.

4. Higher cost for culture

5. Can express large proteins (50 kb )

REFERENCES :-1. DNA ANALYSIS & GENE CLONING BY T.A BROWN

2. PRINCIPLES OF GENETIC MANIPULATION BY S.B PRIMROSE

3. MOLECULAR BIOTECHNOLOGY BY GLICK & PASTERNAK

4. BIOTECHNOLOGY BY B.D SINGH

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