1. Recombinant protein expression in E.coli
Bio
Vigdis Lauvrak

2. 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

3. 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

4. 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?

5. A replicom is a genetic unit consisting of an origin of DNA replication and its associated elements.
origin Replicon copy number
pBR322 pMB
colE1 colE
pUC mod pMB
pMOB45 pKN
pACYC p15A
pSC101 pSC101 5

6. 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) : copies (may be unstable without a selectable marker).
Tn1545 site specific recombination (Peredelchuck and Bennett 1997) - time consuming

7. 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

8. Basic elements of E.coli expression systems
R P SD coding sequence TT
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

9. 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)

10. 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

11. 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.

12. 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

13. 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

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

15. 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,

16. 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.

17. 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.

18. 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

22. 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