The discovery of chemical chaperones’ effect on protein folding led to periplasmic protein expression, especially for ones that require an oxidative environment to form disulfide bonds for proper folding. Folding of proteins that are difficult to do in the cytoplasm can be enhanced in the periplasm where the osmotic pressure can be readily controlled. The osmotic pressure of the periplasmic space can be simply altered by changing that of the medium as osmolytes freely penetrate the outer membrane.