Download Recombinant protein expression in E.coli

Recombinant protein
expression in E.coli
Bio4600 2003
Vigdis Lauvrak
Modern Biotechnology- enabling technologies
Protein
technologies
Computational
technologies
Bioinformatics
Folding Prediction
Docking
Homology Modelling
Cloning
Expression
Purification
Molecular Evolution
Interaction Maps
Structural Biology
Crystallization
Data Collection
Structure Determination
Host: E.coli
Vector: Plasmid
A tremendous number of modified strains
A tremendous number of highly specialized
constructs
Growth medium
Selectable
marker
gene
E.coli
replicon
Promoter
Plasmid
DNA
Cytoplasma
Cloningsites
Leader sequences
Genomic
DNA
Tags
Tags
Gene to be
expressed
Periplasma
Inner membrane
Outer membrane
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
pBR322
colE1
pUC
pMOB45
pACYC
pSC101
Replicon
pMB1
colE1
mod pMB1
pKN402
p15A
pSC101
copy number
15-20
15-20
500-700
15-118
18-22
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
-35
SD
coding sequence
-10
TTGACA(N)17TATAAT
TT
STOP codon
START codon
mRNAUAAGGAGG(N)8AUG (91%)
GUG (8%)
UUG (1%)
R: Reprossor
P: Promoter
SD: Shine Delgarno sequence
(Ribosome binding site- start of mRNA)
(TT: terminator (stabilizes mRNA))
UAU
UGA
UAG
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
lacI operon
Pl
lacI
lac repressor
Pl
lacI
lac Operon
Plac lacO
lacZ
lacY
Beta-galactosidase
(cleavage of lactose)
Plac lacO
lacZ
Beta galpermease
(import of lactose)
lacY
lacA
Beta galtransacetylase
(function?)
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.
IPTG
Pl
lacI
PX lacO
Pl
lacI
PX lacO
The CI binding site (lac operator) can be combinde with various
other promoter sequences to give improved regulation.
Genomic
DNA
Plasmid
DNA
Direct control:
Plac/PI may directly control the production
of plasmid encoded heterologous protein:
Plac
Heterologous protein
Indirect control:
PI lacI
A regualtory protein under lacI
control
Plac
The lac repressor may
be under control of PI
in the genome or on
the plasmid (lacIE.coli).
PX
regulatory protein
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
Growth medium
Plasmid
DNA
Cytoplasma
Genomic
DNA
Periplasma
Inner membrane
Outer membrane
Cytoplasmic expression
Advantages:
•No need for signal sequences,
•High concentration of expressed
protein
Disadvantages:
•Formation of inclusion bodies
•(No disulfide bond formation),
•Protein instability,
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