title: Gene fragment polymerization gives increased yields of recombinant human proinsulin C-peptide.
abstract: A multimerization strategy to improve yields upon recombinant production of the 31-aa human proinsulin C-peptide is presented. Gene fragments encoding the C-peptide were assembled using specific head-to-tail multimerization. DNA constructs encoding one, three or seven copies of the C-peptide gene, fused to a serum albumin binding affinity tag, were expressed intracellularly in Escherichia coli. The three fusion proteins were produced at similar levels (approximately 50 mg/l) and were proteolytically stable during production. Enzymatic digestion by trypsin-carboxypeptidase B treatment of the fusion proteins was shown to efficiently release native C-peptide, as determined by mass spectrometry, reverse-phase chromatography and a radioimmunoassay. The quantitative yields of C-peptide obtained from the three different fusion proteins suggest that this multimerization strategy could provide a cost-efficient production scheme for the C-peptide, and that this strategy could be useful also for production of other recombinant peptides.
authors.affiliation: Department of Biochemistry and Biotechnology, Kungliga Tekniska Högskolan, Stockholm, Sweden.
authors.forename: P
authors.initials: P
authors.lastname: Jonasson
authors.forename: P A
authors.initials: PA
authors.lastname: Nygren
authors.forename: B L
authors.initials: BL
authors.lastname: Johansson
authors.forename: J
authors.initials: J
authors.lastname: Wahren
authors.forename: M
authors.initials: M
authors.lastname: Uhlén
authors.forename: S
authors.initials: S
authors.lastname: Ståhl
mesh_terms: D000595:Amino Acid Sequence; D002096:C-Peptide / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D002096:C-Peptide; D011993:Recombinant Fusion Proteins; D004247:DNA; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D014357:Trypsin