Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm

Under physiological conditions, the Escherichia coli cytoplasm is maintaine d in a reduced state that strongly disfavors the formation of stable disulf ide bonds in proteins. However, mutants in which the reduction of both thio redoxins and glutathione is impaired (trxB gor mutants) accumulate oxidized , enzymatically active alkaline phosphatase in the cytoplasm. These mutants grow very poorly in the absence of an exogenous reductant and accumulate e xtragenic suppressors at a high frequency. One such suppressor strain, FA11 3, grows almost as rapidly as the wild type in the absence of reductant, ex hibits slightly faster kinetics of disulfide bond formation, and has fully induced activity of the transcriptional activator, OxyR. FA113 gave substan tially higher yields of properly oxidized proteins compared with wild-type or trxB mutant strains. For polypeptides with very complex patterns of disu lfide bonds, such as vtPA and the full-length tPA, the amount of active pro tein was further enhanced up to 15-fold by coexpression of TrxA (thioredoxi n 1) mutants with different redox potentials, or 20-fold by the protein dis ulfide isomerase, DsbC. Remarkably, higher yields of oxidized, biologically active proteins were obtained by expression in the cytoplasm of E. coli FA 113 compared with what could be achieved via secretion into the periplasm o f a wild-type strain, even under optimized conditions. These results demons trate that the cytoplasm can be rendered sufficiently oxidizing to allow ef ficient formation of native disulfide bonds without compromising cell viabi lity.