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Functionalized silk assembled from a recombinant spider silk fusion protein (Z-4RepCT) produced in the methylotrophic yeast Pichia pastoris.
Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Uppsala, Sweden.
Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Uppsala, Sweden.; Swedish Univ Agr Sci, Dept Chem & Biotechnol, Uppsala, Sweden.
Univ Tokyo, Grad Sch Agr & Life Sci, Dept Biomat Sci, Tokyo, Japan.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
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2016 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 11, no 5, 687-699 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2016. Vol. 11, no 5, 687-699 p.
National Category
Natural Sciences
URN: urn:nbn:se:uu:diva-287507DOI: 10.1002/biot.201500412ISI: 000375078600011PubMedID: 26814048OAI: oai:DiVA.org:uu-287507DiVA: diva2:922853
VINNOVASwedish Research Council, VR-NT 2013-6104Knut and Alice Wallenberg FoundationSwedish Research Council Formas, 2013-883Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2016-04-25 Created: 2016-04-25 Last updated: 2016-06-23Bibliographically approved

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Ramström, Margareta
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Analytical ChemistryScience for Life Laboratory, SciLifeLab
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