title: Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellular heptameric fusion protein.
abstract: An integrated bioprocess has been developed suitable for production of recombinant peptides using a gene multimerization strategy and site-specific cleavage of the resulting gene product. The process has been used for production in E. coli of the human proinsulin C-peptide via a fusion protein BB-C7 containing seven copies of the 31-residues C-peptide monomer. The fusion protein BB-C7 was expressed at high level, 1.8 g l(-1), as a soluble gene product in the cytoplasm. A heat treatment procedure efficiently released the BB-C7 fusion protein into the culture medium. This step also served as an initial purification step by precipitating the majority of the host cell proteins, resulting in a 70% purity of the BB-C7 fusion protein. Following cationic polyelectrolyte precipitation of the nucleic acids and anion exchange chromatography, native C-peptide monomers were obtained by enzymatic cleavage at flanking arginine residues. The released C-peptide material was further purified by reversed-phase chromatography and size exclusion chromatography. The overall yield of native C-peptide at a purity exceeding 99% was 400 mg l(-1) culture, corresponding to an overall recovery of 56%. The suitability of this process also for the production of other recombinant proteins is discussed.
authors.affiliation: Department of 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: H
authors.initials: H
authors.lastname: Jörnvall
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: D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D002096:C-Peptide / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000737:chemistry / Q000378:metabolism; D011232:Chemical Precipitation; D002853:Chromatography, Liquid / Q000379:methods; D006358:Hot Temperature; D006801:Humans; D009696:Nucleic Acids / Q000302:isolation & purification; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
chemical_list: D002096:C-Peptide; D009696:Nucleic Acids; D011993:Recombinant Fusion Proteins; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D014357:Trypsin