title: Functionalized silk assembled from a recombinant spider silk fusion protein (Z-4RepCT) produced in the methylotrophic yeast Pichia pastoris.
abstract: 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.
authors.affiliation: Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: Ronnie
authors.initials: R
authors.lastname: Jansson
authors.affiliation: Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.|Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: Cheuk H
authors.initials: CH
authors.lastname: Lau
authors.affiliation: Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Ishida
authors.affiliation: Department of Chemistry (BMC) and Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Margareta
authors.initials: M
authors.lastname: Ramström
authors.affiliation: Department of Chemistry and Biotechnology, Swedish University of Agricultural Sciences, Uppsala, Sweden. mats.sandgren@slu.se.
authors.forename: Mats
authors.initials: M
authors.lastname: Sandgren
authors.affiliation: Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden. myh@kth.se.|Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden. myh@kth.se.
authors.forename: My
authors.initials: M
authors.lastname: Hedhammar
mesh_terms: D000818:Animals; D001665:Binding Sites; D005818:Genetic Engineering; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D000072417:Protein Domains; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D047011:Silk / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013112:Spiders / Q000235:genetics* / Q000378:metabolism; D013696:Temperature
chemical_list: D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D047011:Silk
keywords: Glycosylation; Heterologous protein expression; Intracellular/extracellular protein; Pichia pastoris; Recombinant spider silk