title: Increased production of human proinsulin in the periplasmic space of Escherichia coli by fusion to DsbA.
abstract: The production of human proinsulin in its disulfide-intact, native form in Escherichia coli requires disulfide bond formation and the periplasmic space is the favourable compartment for oxidative folding. However, the secretory expression of proinsulin is limited by its high susceptibility to proteolysis and by disulfide bond formation, which is rate-limiting for proinsulin folding. In this report we describe a method for the production of high amounts of soluble, native human proinsulin in E. coli. We fused proinsulin to the C-terminus of the periplasmic disulfide oxidoreductase DsbA via a trypsin cleavage site. As DsbA is the main catalyst of disulfide bond formation in E. coli, we expected increased yields of proinsulin by intra- or intermolecular catalysis of disulfide bond formation. In the context of the fusion protein, proinsulin was found to be stabilised, probably due to an increased solubility and faster disulfide bond formation. To increase the yield of DsbA-proinsulin in the periplasm, several parameters were optimised, including host strains and cultivation conditions, and in particular growth medium composition and supplement of low molecular weight additives. We obtained a further, about three-fold increase in the amount of native DsbA-proinsulin by addition of L-arginine or ethanol to the culture medium. The maximum yield of native human proinsulin obtained from the soluble periplasmic fraction after specific cleavage of the fusion protein with trypsin was 9.2 mg g(-1), corresponding to 1.8% of the total cell protein.
authors.affiliation: Martin-Luther-Universität Halle-Wittenberg, Institut für Biotechnologie, Kurt-Mothes-Str. 3, D-06120, Halle, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Winter
authors.forename: P
authors.initials: P
authors.lastname: Neubauer
authors.forename: R
authors.initials: R
authors.lastname: Glockshuber
authors.forename: R
authors.initials: R
authors.lastname: Rudolph
mesh_terms: D001120:Arginine / Q000378:metabolism; D001709:Biotechnology / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D006801:Humans; D008903:Minerals / Q000494:pharmacology; D010957:Plasmids; D011384:Proinsulin / Q000235:genetics*; D019704:Protein Disulfide-Isomerases / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012492:Salts / Q000494:pharmacology; D013696:Temperature
chemical_list: D008903:Minerals; D011993:Recombinant Fusion Proteins; D012492:Salts; D011384:Proinsulin; D001120:Arginine; D019704:Protein Disulfide-Isomerases