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Integrated bioproce...
Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellular heptameric fusion protein
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Jonasson, P. (författare)
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- Nygren, Per-Åke (författare)
- KTH,Bioteknologi
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Jornvall, H. (författare)
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Johansson, B. L. (författare)
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Wahren, J. (författare)
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Uhlen, M. (författare)
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- Ståhl, Stefan (författare)
- KTH,Bioteknologi
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(creator_code:org_t)
- 2000
- 2000
- Engelska.
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Ingår i: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 76:03-feb, s. 215-226
- Relaterad länk:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- 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.
Nyckelord
- 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
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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