Production in Pichia pastoris of complementary protein-based polymers with heterodimer-forming WW and PPxY domains.
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.
Wageningen UR Food and Biobased Research, Bornse Weilanden 9, 6708 WG, Wageningen, The Netherlands.|Physical Chemistry and Soft Matter, Wageningen University, Stippeneng 4, 6708 WE, Wageningen, The Netherlands.
Natalia E
NE
Domeradzka
Wageningen UR Food and Biobased Research, Bornse Weilanden 9, 6708 WG, Wageningen, The Netherlands. marc.werten@wur.nl.
Marc W T
http://orcid.org/0000-0002-7412-0000
MW
Werten
Physical Chemistry and Soft Matter, Wageningen University, Stippeneng 4, 6708 WE, Wageningen, The Netherlands.
Renko
R
de Vries
Wageningen UR Food and Biobased Research, Bornse Weilanden 9, 6708 WG, Wageningen, The Netherlands.
Frits A
FA
de Wolf
D000595:Amino Acid Sequence; D002151:Calorimetry; D019281:Dimerization; D006031:Glycosylation; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000032:analysis; D010766:Phosphorylation; D010843:Pichia / Q000378:metabolism*; D011108:Polymers / Q000737:chemistry / Q000378:metabolism*; D011392:Proline / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D008024:Ligands; D010455:Peptides; D011108:Polymers; D011994:Recombinant Proteins; D011392:Proline
Heterodimerization; O-glycosylation; PPxY domain; Phosphorylation; Pichia pastoris; Protein coupling; Protein-based polymers; WW domain