Vector
Host
Growth Conditions
Thus, you should consider the solutions for YOUR expression problems at the levels of vector, strains and induction conditions.
Initiation problems
Rare codons
Your gene induces rearrangement and loss of the DE3 lysogen RNA degradation
Tightly suppress gene expression prior to induction Use low-copy origin of replication plasmid Change vector to structured RNA vector
Aggregation
Reason Protein is misfolded due to lack of correct disulfide bond formation Hydrophobic protein Vector Use thioredoxin, DsbA, DsbC fusion partners Clone in a vector containing secretion signal to the periplasm (pelB, OmpA) Solubility enhancing fusion proteins (MBP, NusA, GST, etc.) Host Strain Use Trx(-)/gor(-) strains (e.g. Origami) for creating oxidative conditions in cytosol Membrane rich strains (C41/C43) Growth Conditions Lowaring induction temperature usually helps
Slow expression rate (low temp; low [inducer]; short induction time; poor media) Heat shock with chemical chaperones Heat shock with chemical chaperones
No appropriate chaperones
Truncated protein
Reason Rare codons Vector Optimize codon usage 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
Sub-clone with another fusion partner or avoid N-terminus fusion protein Detect and replace specific protease sites