2. Protein Expression in Bacteria
1. Advantages/disadvantages
2. Genetic elements essential for the expression
3. Cloning strategies
4. Overview of the available expression systems
and expression strains
5. Design of cloning procedures using the VNTI
program
4. Disadvantages
Limitation for expression of eukaryotic proteins due to:
•different frequencies with which the different codons appear in
genes of these organisms
E.g. CGT, CGC, CGG, AGG, AGA, CGA code for arginine, but the last 3
(AGG, AGA, CGA) are rarely used in E. coli and it has low amounts of
respective tRNAs.
•differences in post-translational modifications (
SS bonds, glycosylation etc)
6. Goals
To obtain
as much as possible /good expression+good cell growth
soluble folded protein /reduced aggregation
in a form that is easy to purify /use of secretion and tags
Common problem:
High expression=danger of aggregation, decreased cell growth
10. Promoters
Host’s promoters
2500 in the entire genome of E. coli K12 strain
Most frequently used: Plac / Ptac / Ptrc, PPBAD, rhaPBAD
- Regulation of expression
Promoters from phages
T7, T3, SP6, T5, PL
- Highly efficient and specific expression
12. Plac, Ptac, Ptrc: Characteristics
Level of expression
(inductor)
Key features
Plac Low level up to
middle (IPTG)
Weak, regulated. Suitable for
expression of gene products at
very low intracellular level.
Comparatively expensive
induction.
Ptac
Ptrc
(trp-lac)
Moderately high
(IPTG)
High-level, but lower than T7
system. Regulated expression still
possible.Comparatively expensive
induction. High basal level.
14. PPBAD and RhaPBAD
Level of expression
(inductor)
Key features
PPBAD Variable from low to
high level
(L-arabinose)
Can fine-tune expression levels in
a dose-dependent manner. Tight
regulation possible. Low basal
level. Inexpensive inducer.
rhaPBAD Variable from low to
high level
(L-rhamnose)
Tight regulation. Low basal
activity. Relatively expensive
inducer.
15. Phage Promoters
Level of expression
(inductor)
Key features
T7
T5
Very high
High
Utilizes T7 RNA polymerase.
Utilizes E. coli RNA polymerase.
PL Moderately high
(temperature shift)
Temperature-sensitive host required.
Less likelihood of "leaky" un-induced
expression. Basal level; high basal
level by temperatures below 30°C.
No inducer.
20. Protein Expression in Bacteria
Part2
1. Cloning strategies
2. Overview of the available expression
systems and expression strains
3. Design of cloning procedures using the
VNTI program
29. Expression of Fusion Proteins
We may fuse the target protein with
• various tags to facilitate its purification or detection
HHHHHH-target, epitope-target
• highly soluble proteins to improve solubility and to
facilitate purification
Thioredoxin-target, GST-target
• signal peptides or other proteins or domains to
promote secretion
SP-target
