Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 506116
Title Production in Pichia pastoris of complementary protein-based polymers with heterodimer-forming WW and PPxY domains
Author(s) Domeradzka, Natalia E.; Werten, Marc W.T.; Vries, Renko de; Wolf, Frits A. de
Source Microbial Cell Factories 15 (2016)1. - ISSN 1475-2859
Department(s) Physical Chemistry and Soft Matter
FBR Bioconversion
Publication type Refereed Article in a scientific journal
Publication year 2016
Keyword(s) Heterodimerization - O-glycosylation - Phosphorylation - Pichia pastoris - PPxY domain - Protein coupling - Protein-based polymers - WW domain

Background: Specific coupling of de novo designed recombinant protein polymers for the construction of precisely structured nanomaterials is of interest for applications in biomedicine, pharmaceutics and diagnostics. An attractive coupling strategy is to incorporate specifically interacting peptides into the genetic design of the protein polymers. An example of such interaction is the binding of particular proline-rich ligands by so-called WW-domains. In this study, we investigated whether these domains can be produced in the yeast Pichia pastoris as part of otherwise non-interacting protein polymers, and whether they bring about polymer coupling upon mixing. Results: We constructed two variants of a highly hydrophilic protein-based polymer that differ only in their C-terminal extensions. One carries a C-terminal WW domain, and the other a C-terminal proline-rich ligand (PPxY). Both polymers were produced in P. pastoris with a purified protein yield of more than 2 g L-1 of cell-free broth. The proline-rich module was found to be O-glycosylated, and uncommonly a large portion of the attached oligosaccharides was phosphorylated. Glycosylation was overcome by introducing a Ser → Ala mutation in the PPxY peptide. Tryptophan fluorescence monitored during titration of the polymer containing the WW domain with either the glycosylated or nonglycosylated PPxY-containing polymer revealed binding. The complementary polymers associated with a Kd of ~3 μM, regardless of glycosylation state of the PPxY domain. Binding was confirmed by isothermal titration calorimetry, with a Kd of ~9 μM. Conclusions: This article presents a blueprint for the production in P. pastoris of protein polymers that can be coupled using the noncovalent interaction between WW domains and proline-rich ligands. The availability of this highly specific coupling tool will hereafter allow us to construct various supramolecular structures and biomaterials.

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