Three major factors influence protein expression

1
Three major factors influence protein expression
Host
Vector
Growth Conditions
Thus, you should consider the solutions for YOUR
expression problems at the levels of vector,
strains and induction conditions.
2
No / Low Protein Production
Growth Conditions Host Strain Vector Reason
Start induction at higher OD Shorten induction time Add Glucose to suppress leaky expression Use BL21AI or BL21(DE3)pLysS/E Use T7 promoter-based vectors (also arabinose) Tightly regulate induction with lac operator Toxic protein
Re-clone with more A residues at 5 Shorten distance between RBS and first ATG (2-8 nt( Initiation problems
Slow translation by reducing temperature or grow in poor media Use stains supplementing rare codons (Rosetta, Codon ) Mutate gene for codon optimization Rare codons
Start from freshly transformed bacteria Add Glucose to suppress leaky expression Use recA- strains (HMS174 BLR) Tightly suppress gene expression prior to induction Use low-copy origin of replication plasmid Your gene induces rearrangement and loss of the DE3 lysogen
Use RNAse deficient strain (BL21Star) Change vector to structured RNA vector RNA degradation
3
Aggregation
Growth Conditions Host Strain Vector Reason
Lowaring induction temperature usually helps Use Trx(-)/gor(-) strains (e.g. Origami) for creating oxidative conditions in cytosol Use thioredoxin, DsbA, DsbC fusion partners Clone in a vector containing secretion signal to the periplasm (pelB, OmpA) Protein is misfolded due to lack of correct disulfide bond formation
Slow expression rate (low temp low inducer short induction time poor media) Heat shock with chemical chaperones Membrane rich strains (C41/C43) Solubility enhancing fusion proteins (MBP, NusA, GST, etc.) Hydrophobic protein
Heat shock with chemical chaperones Screen various expressing strains Add vectors for various chaperone co-expression No appropriate chaperones
Induce at low temp. Membrane rich (C41/C43) Replace with bacterial signals (secretion) or omit signals Sub-cellular localization signals
Membrane rich (C41/C43) Use mistic fusion protein. Generate truncated forms of protein (soluble domains) Membrane proteins
Heat shock with chemical chaperones Transform with a partner combination of 2-4 vectors for max 8 proteins Protein is part of a complex
4
Truncated protein
Growth Conditions Host Strain Vector Reason
Slow elongation by low temp. low inducer poor media Use rare codon strains (rosetta , codonPlus) Optimize codon usage Rare codons
Slow expression rate with low temp. low inducer short harvest poor media Sub-clone with another fusion partner or avoid N-terminus fusion protein Faster, uncoordinated-translation of fusion protein
Grow and induce at low temp, use protease inhibitors when breaking the cells on ice Induce at higher OD and reduce induction time Low protease strains (BL21 derivatives, M15) Detect and replace specific protease sites Degradation