Recombinant protein expression in E.coli Bio4600 2003 Vigdis Lauvrak

Modern Biotechnology- enabling technologies Protein technologies Computational technologies Cloning Expression Purification Molecular Evolution Interaction Maps Bioinformatics Folding Prediction Docking Homology Modelling Structural Biology Crystallization Data Collection Structure Determination

Vector: Plasmid Host: E.coli E.coli replicon Growth medium A tremendous number of highly specialized constructs Host: E.coli A tremendous number of modified strains E.coli replicon Promoter Cytoplasma Periplasma Growth medium Inner membrane Outer membrane Genomic DNA Plasmid DNA Selectable marker gene Cloningsites Leader -sequences Tags Tags Gene to be expressed

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 number pBR322 pMB1 15-20 colE1 colE1 15-20 pUC mod pMB1 500-700 pMOB45 pKN402 15-118 pACYC p15A 18-22 pSC101 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 inserts Increased 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

Basic elements of E.coli expression systems R P SD coding sequence TT -35 -10 STOP codon TTGACA(N)17TATAAT START codon UAU mRNAUAAGGAGG(N)8AUG (91%) UGA GUG (8%) UAG UUG (1%) R: Reprossor P: Promoter SD: Shine Delgarno sequence (Ribosome binding site- start of mRNA) (TT: terminator (stabilizes mRNA)) 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)

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

How does it work? Pl lacI Plac lacO lacZ lacY lacA Pl lacI Plac lacO lacZ lacY lacA 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.

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. IPTG Pl lacI PX lacO Pl lacI PX lacO

Direct control: Indirect control: Genomic DNA Plasmid DNA Direct control: Plac/PI may directly control the production of plasmid encoded heterologous protein: Plac Heterologous protein Indirect control: A regualtory protein under lacI control PI lacI Plac regulatory protein The lac repressor may be under control of PI in the genome or on the plasmid (lacI- E.coli). PX Heterologous protein

The pET 11 vectors (Novagen and Stratagene) with T7/lacO promoter : 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 PI lacI A copy of the lacI gene (also found in the genome) is inserted on the plasmids to achieve sufficient repressor.

Choice of E.coli compartment Cytoplasma Periplasma Growth medium Inner membrane Outer membrane Genomic DNA Plasmid DNA Cytoplasmic expression Advantages: No need for signal sequences, High concentration of expressed protein Disadvantages: Formation of inclusion bodies (No disulfide bond formation), Protein instability,

No efficient system for direct transport to growth media. Periplasm Advantages 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 media No 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