title: Overexpression of Escherichia coli oxidoreductases increases recombinant insulin-like growth factor-I accumulation.
abstract: Transient overexpression of either DsbA or DsbC can double the yield of periplasmic insulin-like growth factor (IGF)-I in Escherichia coli to 8.5 g/liter. Strikingly, most of the overexpressed DsbA or DsbC is found in the reduced form, implying that enhanced disulfide isomerization is responsible for the substantial increase in IGF-I yield. All of the accumulated IGF-I has had the signal sequence removed, underscoring the secretion capacity of this organism as well as its utility for efficient production of polypeptide with the correct amino terminus. The overexpressed IGF-I constitutes approximately 30% of the total cell protein. Overproduction of active site mutants of DsbA instead of the wild-type gene do not produce this increase in yield. With wild-type levels of DsbA and DsbC, most of the secreted IGF-I is found in disulfide-linked aggregates, although 10% is soluble and about 5% is correctly folded. Contrary to expectations, overexpression of the disulfide oxidoreductases decreased the soluble fraction. Because the aggregated protein can be efficiently solubilized and refolded, the increased yield is a significant benefit for the production of IGF-I.
authors.affiliation: Department of Cell Culture and Fermentation, Research and Development, Genentech, Inc., South San Francisco, CA 94080, USA. joly@gene.com
authors.forename: J C
authors.initials: JC
authors.lastname: Joly
authors.forename: W S
authors.initials: WS
authors.lastname: Leung
authors.forename: J R
authors.initials: JR
authors.lastname: Swartz
mesh_terms: D001709:Biotechnology / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis* / Q000235:genetics; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*
chemical_list: D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D019704:Protein Disulfide-Isomerases