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Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellular heptameric fusion protein
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0003-4214-6991
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2000 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 76, no 03-feb, 215-226 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2000. Vol. 76, no 03-feb, 215-226 p.
Keyword [en]
fusion protein, multimerization, enzymatic cleavage, heat release, C-peptide, Escherichia coli, expanded-bed adsorption, aqueous 2-phase systems, thermostable dna-polymerase, respiratory syncytial virus, single-step purification, escherichia-coli, renal-function, engineering proteins, recombinant protein, human insulin
URN: urn:nbn:se:kth:diva-19496DOI: 10.1016/S0168-1656(99)00195-9ISI: 000084886400011OAI: diva2:338188
QC 20100525Available from: 2010-08-10 Created: 2010-08-10Bibliographically approved

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Nygren, Per-ÅkeStåhl, Stefan
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