title: High-level C5a gene expression and recovery of recombinant human C5a from Escherichia coli.
abstract: Poor expression of a synthetic gene for the inflammatory mediator, C5a, was observed in E. coli grown in rich media. Varying the media composition markedly improved expression, although C5a levels still declined rapidly at the end of log phase. Using a protease-deficient strain, C5a was recovered at stationary phase in high yield (13 mg/liter of culture). Recovery was dependent on guanidinium hydrochloride extraction to solubilize the protein and glutathione treatment to promote correct folding. Two-thirds of the C5a retained an amino-terminal methionine. Both forms of recombinant C5a had activity similar to serum-derived C5a in binding to human neutrophil receptors and inducing chemotaxis. The 700-fold improvement in yield made it feasible to obtain gram amounts of C5a and provides an efficient system for site-directed mutagenesis.
authors.affiliation: Immunoscience Research Area, Abbott Laboratories, Abbott Park, Illinois 60064.
authors.forename: K W
authors.initials: KW
authors.lastname: Mollison
authors.forename: T A
authors.initials: TA
authors.lastname: Fey
authors.forename: R A
authors.initials: RA
authors.lastname: Krause
authors.forename: W
authors.initials: W
authors.lastname: Mandecki
authors.forename: J L
authors.initials: JL
authors.lastname: Fox
authors.forename: G W
authors.initials: GW
authors.lastname: Carter
mesh_terms: D003001:Cloning, Molecular; D003182:Complement C5 / Q000096:biosynthesis / Q000235:genetics*; D015936:Complement C5a; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D003182:Complement C5; D011994:Recombinant Proteins; D015936:Complement C5a