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Functionalized silk...
Functionalized silk assembled from a recombinant spider silk fusion protein (Z-4RepCT) produced in the methylotrophic yeast Pichia pastoris
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- Jansson, Ronnie (author)
- Swedish University of Agricultural Sciences,Sveriges lantbruksuniversitet,Institutionen för anatomi, fysiologi och biokemi,Department of Anatomy, Physiology and Biochemistry (AFB)
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- Lau, Cheuk H. (author)
- Swedish University of Agricultural Sciences,Sveriges lantbruksuniversitet,Institutionen för anatomi, fysiologi och biokemi,Institutionen för Kemi och Bioteknologi,Department of Anatomy, Physiology and Biochemistry (AFB),The Department of Chemistry and Biotechnology
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- Ishida, Takuya (author)
- Univ Tokyo, Grad Sch Agr & Life Sci, Dept Biomat Sci, Tokyo, Japan,University of Tokyo
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- Ramström, Margareta (author)
- Uppsala universitet,Analytisk kemi,Science for Life Laboratory, SciLifeLab
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- Sandgren, Mats (author)
- Swedish University of Agricultural Sciences,Sveriges lantbruksuniversitet,Institutionen för Kemi och Bioteknologi,The Department of Chemistry and Biotechnology
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- Hedhammar, My (author)
- Sveriges lantbruksuniversitet,KTH,Proteinteknologi,Swedish University of Agricultural Sciences, Sweden,Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Uppsala, Sweden.; KTH Royal Inst Technol, Sch Biotechnol, Div Prot Technol, Stockholm, Sweden,Institutionen för anatomi, fysiologi och biokemi,Department of Anatomy, Physiology and Biochemistry (AFB),Royal Institute of Technology (KTH)
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(creator_code:org_t)
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- 2016-03-04
- 2016
- English.
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In: Biotechnology Journal. - : Wiley-VCH Verlagsgesellschaft. - 1860-6768 .- 1860-7314. ; 11:5, s. 687-699
- Related links:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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https://urn.kb.se/re...
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https://res.slu.se/i...
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Abstract
Subject headings
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- Functional biological materials are a growing research area with potential applicability in medicine and biotechnology. Using genetic engineering, the possibility to introduce additional functions into spider silk-based materials has been realized. Recently, a recombinant spider silk fusion protein, Z-4RepCT, was produced intracellularly in Escherichia coli and could after purification self-assemble into silk-like fibers with ability to bind antibodies via the IgG-binding Z domain. In this study, the use of the methylotrophic yeast Pichia pastoris for production of Z-4RepCT has been investigated. Temperature, pH and production time were influencing the amount of soluble Z-4RepCT retrieved from the extracellular fraction. Purification of secreted Z-4RepCT resulted in a mixture of full-length and degraded silk proteins that failed to self-assemble into fibers. A position in the C-terminal domain of 4RepCT was identified as being subjected to proteolytic cleavage by proteases in the Pichia culture supernatant. Moreover, the C-terminal domain was subjected to glycosylation during production in P. pastoris. These observed alterations of the CT domain are suggested to contribute to the failure in fiber assembly. As alternative approach, Z-4RepCT retrieved from the intracellular fraction, which was less degraded, was used and shown to retain ability to assemble into silk-like fibers after enzymatic deglycosylation.
Subject headings
- TEKNIK OCH TEKNOLOGIER -- Industriell bioteknik (hsv//swe)
- ENGINEERING AND TECHNOLOGY -- Industrial Biotechnology (hsv//eng)
- TEKNIK OCH TEKNOLOGIER -- Industriell bioteknik -- Bioprocessteknik (hsv//swe)
- ENGINEERING AND TECHNOLOGY -- Industrial Biotechnology -- Bioprocess Technology (hsv//eng)
- TEKNIK OCH TEKNOLOGIER -- Industriell bioteknik -- Biomaterial (hsv//swe)
- ENGINEERING AND TECHNOLOGY -- Industrial Biotechnology -- Bio Materials (hsv//eng)
- TEKNIK OCH TEKNOLOGIER -- Industriell bioteknik -- Biokatalys och enzymteknik (hsv//swe)
- ENGINEERING AND TECHNOLOGY -- Industrial Biotechnology -- Biocatalysis and Enzyme Technology (hsv//eng)
- NATURVETENSKAP -- Biologi -- Mikrobiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Microbiology (hsv//eng)
Keyword
- Glycosylation
- Heterologous protein expression
- Intracellular/extracellular protein
- Pichia pastoris
- Recombinant spider silk
- Bins
- Biological materials
- Enzyme activity
- Escherichia coli
- Fibers
- Genetic engineering
- Purification
- Recombinant proteins
- Yeast
- C-terminal domains
- Culture supernatant
- Deglycosylation
- Heterologous proteins
- Methylotrophic yeasts
- Proteolytic cleavage
- Spider silks
- Proteins
Publication and Content Type
- ref (subject category)
- art (subject category)
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