Transcript Three Major factors influence protein expression

Three major factors influence protein expression
Vector
Host
Growth Conditions
Thus, you should consider the solutions for YOUR
expression problems at the levels of vector,
strains and induction conditions.
No / Low Protein Production
Reason
Vector
Toxic protein
•Use T7 promoter-based
vectors (also arabinose)
•Tightly regulate induction
with lac operator
Initiation
problems
•Re-clone with more A
residues at 5’
•Shorten distance between
RBS and first ATG (2-8 nt(
Host Strain
Growth Conditions
•Use BL21AI or
BL21(DE3)pLysS/E
•Start induction at higher
OD
•Shorten induction time
•Add Glucose to suppress
leaky expression
Rare codons
Mutate gene for codon
optimization
Use stains
supplementing rare
codons (Rosetta,
Codon +)
Slow translation by
reducing temperature or
grow in poor media
Your gene
induces
rearrangement
and loss of the
DE3 lysogen
•Tightly suppress gene
expression prior to
induction
•Use low-copy origin of
replication plasmid
•Use recA- strains
(HMS174; BLR)
•Start from freshly
transformed bacteria
•Add Glucose to suppress
leaky expression
RNA
degradation
Change vector to
structured RNA vector
Use RNAse deficient
strain (BL21Star)
Aggregation
Reason
Vector
Host Strain
Growth Conditions
Protein is
misfolded due to
lack of correct
disulfide bond
formation
Use thioredoxin, DsbA, DsbC
fusion partners
Clone in a vector containing
secretion signal to the
periplasm (pelB, OmpA)
Use Trx(-)/gor(-)
strains (e.g.
Origami) for
creating oxidative
conditions in
cytosol
Lowaring induction
temperature usually helps
Hydrophobic
protein
Solubility enhancing fusion
proteins (MBP, NusA, GST,
etc.)
Membrane rich
strains
(C41/C43)
Slow expression rate (low
temp; low [inducer]; short
induction time; poor media)
Heat shock with
chemical chaperones
No appropriate
chaperones
Add vectors for various
chaperone co-expression
Screen various
expressing
strains
Heat shock with
chemical chaperones
Sub-cellular
localization
signals
Replace with bacterial
signals (secretion) or omit
signals
Membrane rich
(C41/C43)
Induce at low temp.
Membrane
proteins
Use mistic fusion protein.
Generate truncated forms of
protein (soluble domains)
Membrane rich
(C41/C43)
Protein is part of
a complex
Transform with a partner :
combination of 2-4 vectors
for max 8 proteins
Heat shock with
chemical chaperones
Truncated protein
Reason
Vector
Rare codons
Optimize codon
usage
Faster,
uncoordinatedtranslation of
fusion protein
Sub-clone with
another fusion
partner or avoid Nterminus fusion
protein
Degradation
Detect and replace
specific protease
sites
Host Strain
Use rare codon
strains (rosetta ,
codonPlus)
Growth Conditions
Slow elongation by low
temp.; low inducer;
poor media
Slow expression rate
with low temp.; low
inducer; short harvest;
poor media
Low protease
strains (BL21
derivatives, M15)
Grow and induce at low
temp, use
protease
inhibitors when
breaking the cells
on ice
Induce at higher OD
and reduce
induction time