5. Protein stability
Proteases of the cell envelope cleave cytoplasmic proteins after the cells are lysed.
To overcome this problem protease mutants are the best options. Protease inhibitors work sometimes but there are a number of proteases for which no inhibitors are known. One example is the DegP (HtrA) protease. The only way out is to use protease mutants.
If nothing helps: mix cell lysate immediately after cell disruption to chromatography material (e.g. anionic exchange resin) which has helped in very tough cases.
If protein is degraded during expression, try short induction times. Often proteins are degraded after overnight growth. Short induction times (2-4 h) can still give high yields (can be up to 30 mg/l culture).