Functionalized silk assembled from a recombinant spider silk fusion protein (Z-4RepCT) produced in the methylotrophic yeast Pichia pastoris

Jansson, RonnieSwedish University of Agricultural Sciences,Sveriges lantbruksuniversitet,Institutionen för anatomi, fysiologi och biokemi,Department of Anatomy, Physiology and Biochemistry (AFB) (author)

QC 20160518
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.

Lau, Cheuk H.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(Swepub:slu)115987 (author)
Ishida, TakuyaUniv Tokyo, Grad Sch Agr & Life Sci, Dept Biomat Sci, Tokyo, Japan,University of Tokyo(Swepub:slu)93565 (author)
Ramström, MargaretaUppsala universitet,Analytisk kemi,Science for Life Laboratory, SciLifeLab(Swepub:uu)maram031 (author)
Sandgren, MatsSwedish University of Agricultural Sciences,Sveriges lantbruksuniversitet,Institutionen för Kemi och Bioteknologi,The Department of Chemistry and Biotechnology(Swepub:slu)49440 (author)
Hedhammar, MySveriges 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)(Swepub:slu)46815 (author)
Sveriges lantbruksuniversitetInstitutionen för anatomi, fysiologi och biokemi (creator_code:org_t)
Sveriges lantbruksuniversitet

In:Biotechnology Journal: Wiley-VCH Verlagsgesellschaft11:5, s. 687-6991860-67681860-7314

By the author/editor: Jansson, Ronnie; Lau, Cheuk H.; Ishida, Takuya; Ramström, Margar ...; Sandgren, Mats; Hedhammar, My

About the subject

ENGINEERING AND TECHNOLOGY: ENGINEERING AND ...; and Industrial Biote ...; and Bioprocess Techn ...

ENGINEERING AND TECHNOLOGY: ENGINEERING AND ...; and Industrial Biote ...; and Bio Materials

ENGINEERING AND TECHNOLOGY: ENGINEERING AND ...; and Industrial Biote ...; and Biocatalysis and ...

NATURAL SCIENCES: NATURAL SCIENCES; and Biological Scien ...; and Microbiology

By the university: Royal Institute of Technology; Uppsala University; Swedish University of Agricultural Sciences

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