There are several options for expressing proteins that require disulfide bonding for proper folding and function. The E. coli strain SHuffle® has been genetically engineered for the cytoplasmic production of disulfide bonded proteins. Genetic deletion of the two (glutaredoxin and thioredoxin) reducing pathways in E. coli has resulted in a mutant strain with diminished capacity to reduce proteins and an increased capacity to oxidize cytoplasmically expressed proteins. Additionally, these cells have been engineered to express the disulfide bond isomerase DsbC in the cytoplasm, further enhancing the capacity and fidelity of disulfide bond formation in this host.
It is also possible to use eukaryotic protein secretion or cell-free expression strategies to achieve proper disulfide bonding of proteins. For example, secretion of a target protein into the oxidative ER environment of yeast, mammalian or insect cells will permit a nascent protein the opportunity to fold and to have access to PDI. Cell-free strategies can also be employed using systems like PURExpress® with Disulfide Bond Enhancer (NEB #E6820) that contains components that improve in vitro formation of disulfide bonds.