Three major factors influence protein expression

Host

Growth Conditions

Vector

Thus, you should consider the solutions for YOUR expression problems at the levels of vector, strains and induction conditions.

No / Low Protein ProductionReason Vector Host Strain Growth Conditions

Toxic protein •Use T7 promoter-based vectors (also arabinose)•Tightly regulate induction with lac operator

•Use BL21AI or BL21(DE3)pLysS/E

•Start induction at higher OD •Shorten induction time•Add Glucose to suppress leaky expression

Initiationproblems

•Re-clone with more A residues at 5’•Shorten distance between RBS and first ATG (2-8 nt(

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)

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

Aggregation

Truncated protein

Reason Vector Host Strain Growth Conditions

Rare codons Optimize codon usage

Use rare codon strains (rosetta , codonPlus)

Slow elongation by low temp.; low inducer; poor media

Faster, uncoordinated-translation of fusion protein

Sub-clone with another fusion partner or avoid N-terminus fusion protein

Slow expression rate with low temp.; low inducer; short harvest; poor media

Degradation Detect and replace specific protease sites

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