Recombinant protein expression in E.coli

Bio4600 2003

Vigdis Lauvrak

Computational technologies

BioinformaticsFolding PredictionDockingHomology Modelling

Structural Biology

CrystallizationData CollectionStructure Determination

Protein technologies

CloningExpressionPurificationMolecular EvolutionInteraction Maps

Modern Biotechnology- enabling technologies

Host: E.coliA tremendous number of modified strains

E.coli replicon

Promoter

Cloningsites

Leader -sequences

Tags

Gene to be expressed

Tags

Selectable marker gene

Vector: PlasmidA tremendous number of highly specialized constructs

Cytoplasma

Periplasma

Growth medium

Inner membrane

Outer membrane

Genomic DNA

Plasmid DNA

Major options to be considered:

•Gene dosage (copy number)•Level of expression•Which compartment to harvest from•Tags for purification, improvement of

stability and solubility•Codon usage E.coli:recombinant protein•Purpose of expression: Large scale

industrial/or analytical levels?

Major options to be considered:

•Gene dosage (copy number)•Level of expression•Which compartment to harvest from•Tags for purification, improvement of

stability and solubility•Codon usage E.coli:recombinant protein•Purpose of expression: Large scale

industrial/or analytical levels?

A replicom is a genetic unit consisting of an origin of DNA replication and its associated elements.

origin Replicon copy numberpBR322 pMB1 15-20colE1 colE1 15-20pUC mod pMB1 500-700pMOB45 pKN402 15-118pACYC p15A 18-22pSC101 pSC101 5

Gene dosage

Medium to high copy number plasmids•Relaxed replication•Random distribution•Relatively low loss: Continously growth and toxic genes/gene products will lead to plasmid loss.

Increased plasmid stability:Selectable markers

•Genes for antibiotic resistance•Complementation: An essential chromosomal gene is deleted or mutated and an intact copy or a supressor is suplied in trans. •Genes or repressors that lead to cell death upon plasmid loss.

Duplication of genomic insertsIncreased gene dosage in E-coli genome:

•RecA dupllication of insert (Olson et al. 1998) : 15--40 copies (may be unstable without a selectable marker). •Tn1545 site specific recombination (Peredelchuck and Bennett 1997) - time consuming

Gene dosage

Medium to high copy number plasmids•Relaxed replication•Random distribution•Relatively low loss: Continously growth and toxic genes/gene products will lead to plasmid loss.

Increased plasmid stability:Selectable markers

•Genes for antibiotic resistance•Complementation: An essential chromosomal gene is deleted or mutated and an intact copy or a supressor is suplied in trans. •Genes or repressors that lead to cell death upon plasmid loss.

Duplication of genomic insertsIncreased gene dosage in E-coli genome:

•RecA dupllication of insert (Olson et al. 1998) : 15--40 copies (may be unstable without a selectable marker). •Tn1545 site specific recombination (Peredelchuck and Bennett 1997) - time consuming

Control of expression level

Desired:

High expression level

(10-30% or more of produced protein)

Observed:

Many proteins may are toxic at high doses.

Solution:

Regulation of expression

Control of expression level

Desired:

High expression level

(10-30% or more of produced protein)

Observed:

Many proteins may are toxic at high doses.

Solution:

Regulation of expression

Basic elements of E.coli expression systems

R: Reprossor

P: Promoter

SD: Shine Delgarno sequence

(Ribosome binding site- start of mRNA)

(TT: terminator (stabilizes mRNA))

-35 -10 STOP codon

TTGACA(N)17TATAAT START codon UAU

mRNAUAAGGAGG(N)8AUG (91%) UGA GUG (8%) UAG UUG (1%)

R P SD coding sequence TT

E.coli expression vectors: contain:

• E.coli expression elements

•Unique cloning sites

•An origin of replication

•A selectable marker

Level of regulation depends on the promoter

•The lac operon- the paradigm of protein regulation in E.coli: lactose/ IPTG-induction (derepression)

•lacUV5 (leaky): IPTG

•tac and trc synthetic versions of lac (tighter): IPTG

•T7-late promoter : Depends on T7 polymerase

•PL promoter- Lambda CI regulated, tight regulation

•cspA: Cold chock induction

•phoA, trp and araBAD (PBAD): Nutritional inducible

•tet: Tetracycline inducible

•Signal dependent promoters: pH, oxygen conc., osmolarity etc. (Inexpensive large scale production)

Level of regulation depends on the promoter

•The lac operon- the paradigm of protein regulation in E.coli: lactose/ IPTG-induction (derepression)

•lacUV5 (leaky): IPTG

•tac and trc synthetic versions of lac (tighter): IPTG

•T7-late promoter : Depends on T7 polymerase

•PL promoter- Lambda CI regulated, tight regulation

•cspA: Cold chock induction

•phoA, trp and araBAD (PBAD): Nutritional inducible

•tet: Tetracycline inducible

•Signal dependent promoters: pH, oxygen conc., osmolarity etc. (Inexpensive large scale production)

Pl lacI Plac lacO lacZ lacY lacA

lacI operon lac Operon

lac repressor Beta-galactosidase Beta gal- (cleavage of lactose) Beta gal- transacetylase permease (function?) (import of lactose)

The lac operon -the paradigm of expression regulation in E.coli

Pl lacI Plac lacO lacZ lacY lacA

In presence of glucose (no starvation/ low cAMP level) the lac repressor (lacI gene product) is bound to the lac operator and blocks RNA polymerase from binding DNA - Thus the lacI geneproduct acts as an repressor (inhibitor of transcription). In the absence

In periods of glucose starvation (high level of cAMP) and presence of lactose:Lactose enetrs the cell and binds to the LacI repressor protein making it fall of the DNA. RNA polymerase can now bind to the lac promoter and initiate transcription.

-Lactose acts as an inducer (by removing the repressor) of transcription.

How does it work?

Pl lacI Plac lacO lacZ lacY lacA

Pl lacI Plac lacO lacZ lacY lacA

The lac promoter of E.coli expression vectors:

• Induction is performed with IPTG which acts as a synthetic lactose analogue that binds the lacI gene product.

•Presence of glucose further prevents transcription from the lac promoter.

The CI binding site (lac operator) can be combinde with various other promoter sequences to give improved regulation.

Pl lacI PX lacO Pl lacI PX lacO

IPTG

Plac Heterologous protein

Indirect control:

A regualtory protein under lacI control

Genomic DNA

Plasmid DNA

Plac regulatory protein

PI lacI

The lac repressor may be under control of PI in the genome or on the plasmid (lacI- E.coli).

Direct control:

Plac/PI may directly control the production of plasmid encoded heterologous protein:

PX Heterologous protein

The pET 11 vectors (Novagen and Stratagene) with T7/lacO promoter :

PI lacI

lacUV5 T7 polymerase

•T7 RNA polymerase in the bacterial chromosome is controled by a lacUV5 promoter.

• The heterologous protein is under control of the T7 promoter.

•The T7 promoter is fused to the plac operator -

•The lac I repressor inhibits expression of T7 polymerase and the heterologous protein.

•IPTG will induce is used for induction.

T7 lacO Heterologous protein T7 terminator

A copy of the lacI gene (also found in the genome) is inserted on the plasmids to achieve sufficient repressor.

Cytoplasmic expressionAdvantages:

•No need for signal sequences,•High concentration of expressed protein

Disadvantages: •Formation of inclusion bodies•(No disulfide bond formation),•Protein instability,

Choice of E.coli compartment

Cytoplasma

Periplasma

Growth medium

Inner membrane

Outer membrane

Genomic DNA

Plasmid DNA

PeriplasmAdvantages

•Improved folding (no inclusion body formation)•Disulfide bridge formation (may be enhanced by the presence of DsbA and DsbB proteins)•Fewer proteins and possible leakage to growth medium may facilitate purification.•Less protein degradation.

Disadvantages.•Low protein concentration due to inefficient transport and small compartment

Solution •Thight regulation of expression•Molecular chaperones (protein specific)•Temperature down shift after induction- less formation of inclusion bodies).

Growth mediaNo efficient system for direct transport to growth media.Leakage from periplasm is often used.

Common problems encountered with E.coli expression system:

The desired protein may be:Unstable, toxic, insoluble, form inclusion bodies, uncorect folded, depend on disulfide bridges, and active only with postranslational modifications : glycosylation, phosphorylation and amidation.

Solutions:Choice of a suitable E.coli strain, tags, fusions and leader sequences can solve many problems including disulfide bridge formation, but proteins that need correct postranslational modifications as underlined above have to be produced in Eucaryotic systems.

Solutions:

•Thight regulation of expression•Coexpression of molecular chaperones (protein specific)•Reduction of rate of protein synthesis (lower growth rate by temperature down shift after induction)•Fusion moiteties may increase folding, solubility and resistance to proteolysis.•Use of protease deficient E.coli strains•Use of thioredoxin reductase (trxB) og glutatione reductase (gor) double mutants may give disulfide bridge formation in cytosol•Periplasmic expression

Characteristics of suitable induction sensitive promotors

•High strength

•Tight regulation

•Simple and cost effective induction:

Basic research:

IPTG (lactose analogue (toxic))

Tetracycline

Thermal

Industrial production of theraeutics: Thermal

Chemical

Nutrional

Characteristics of suitable induction sensitive promotors

•High strength

•Tight regulation

•Simple and cost effective induction:

Basic research:

IPTG (lactose analogue (toxic))

Tetracycline

Thermal

Industrial production of theraeutics: Thermal

Chemical

Nutrional