Table 2.

Strategies for overcoming common problems during recombinant protein expression in E. coli.

Problem	Possible explanation	Solutions
No or low expression	Protein may be toxic before induction	Control basal induction:
		• add glucose when using expression vectors containing lac-based promoters
		• use defined media with glucose as source of carbon
		• use pLysS/pLysE bearing strains in T7-based systems
		• use promoters with tighter regulation
		Lower plasmid copy number
	Protein may be toxic after induction	Control level of induction:
		• Tuneable promoters
		• Use strains that allow control of induction [Lemo21(DE3) strain] or lacY- strains (TunerTM)
		Lower plasmid copy number
		Use strains that are better for the expression of toxic proteins (C41 or C43)
		Direct protein to the periplasm
	Codon bias	Optimize codon frequency in cDNA to better reflect the codon usage of the host
		Use codon bias-adjusted strains
		Increase biomass:
		• Try new media formulations
		• Provide good aeration and avoid foaming
Inclusion body formation	Incorrect disulfide bond formation	Direct protein to the periplasm
		Use E. coli strains with oxidative cytoplasmic environment
	Incorrect folding	Co-express molecular chaperones
		Supplement media with chemical chaperones and cofactors
		Remove inducer and add fresh media
		Lower production rate:
		• Lower temperature. If possible, use strains with cold-adapted chaperones
		• Tune inducer concentration
	Low solubility of the protein	Fuse desired protein to a solubility enhancer (fusion partners)
	An essential post translational modification is needed	Change microorganism
Protein inactivity	Incomplete folding	Lower temperature
		Monitor disulfide bond formation and allow further folding in vitro
	Mutations in cDNA	Sequence plasmid before and after induction. If mutations are detected, the protein may be toxic.
		Use a recA- strain to ensure plasmid stability
		Transform E. coli before each expression round