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Abstract
Yeast Pichia pastoris is a widely used system for heterologous protein expression. However, post-translational modifications, especially glycosylation, usually impede pharmaceutical application of recombinant proteins because of unexpected alterations in protein structure and function. The aim of this study was to identify glycosylation sites on recombinant human platelet-derived growth factor-BB (rhPDGF-BB) secreted by P. pastoris, and investigate possible effects of O-linked glycans on PDGF-BB functional activity. PDGF-BB secreted by P. pastoris is very heterogeneous and contains multiple isoforms. We demonstrated that PDGF-BB was O-glycosylated during the secretion process and detected putative O-glycosylation sites using glycosylation staining and immunoblotting. By site-directed mutagenesis and high-resolution LC/MS analysis, we, for the first time, identified two threonine residues at the C-terminus as the major O-glycosylation sites on rhPDGF-BB produced in P. pastoris. Although O-glycosylation resulted in heterogeneous protein expression, the removal of glycosylation sites did not affect rhPDGF-BB mitogenic activity. In addition, the unglycosylated PDGF-BBΔGly mutant exhibited the immunogenicity comparable to that of the wild-type form. Furthermore, antiserum against PDGF-BBΔGly also recognized glycosylated PDGF-BB, indicating that protein immunogenicity was unaltered by glycosylation. These findings elucidate the effect of glycosylation on PDGF-BB structure and biological activity, and can potentially contribute to the design and production of homogeneously expressed unglycosylated or human-type glycosylated PDGF-BB in P. pastoris for pharmaceutical applications.
Highlights
The yeast Pichia pastoris has been successfully used for the production of recombinant proteins because it combines the advantages of prokaryotic and mammalian cell expression systems [1].PLOS ONE | DOI:10.1371/journal.pone.0145419 December 23, 2015Glycosylation in Pichia pastorisThe most important advantage over bacterial systems is that yeasts are able, like mammalian cells, to express eukaryotic proteins requiring post-translational modifications (PTMs) such as proteolytic cleavage, glycosylation, and disulfide bond formation [1,2,3,4].Glycosylation is a very common PTM: it is estimated that 50–70% of human proteins are glycosylated [5]
The 104-amino acid fragment was cloned into the pMEX9k vector, where rhPDGF-B expression was controlled by the alcohol oxidase 1 (AOX1) promoter inducible by methanol
The glycosylation status of rhPDGF-BB was examined in the reaction with PNGase F, which hydrolyzes N-linked glycans [28]; rhIFN-ω, an N-glycosylated protein expressed in P. pastoris, was used as a control
Summary
The yeast Pichia pastoris has been successfully used for the production of recombinant proteins because it combines the advantages of prokaryotic and mammalian cell expression systems [1].PLOS ONE | DOI:10.1371/journal.pone.0145419 December 23, 2015Glycosylation in Pichia pastorisThe most important advantage over bacterial systems is that yeasts are able, like mammalian cells, to express eukaryotic proteins requiring post-translational modifications (PTMs) such as proteolytic cleavage, glycosylation, and disulfide bond formation [1,2,3,4].Glycosylation is a very common PTM: it is estimated that 50–70% of human proteins are glycosylated [5]. The yeast Pichia pastoris has been successfully used for the production of recombinant proteins because it combines the advantages of prokaryotic and mammalian cell expression systems [1]. The most important advantage over bacterial systems is that yeasts are able, like mammalian cells, to express eukaryotic proteins requiring post-translational modifications (PTMs) such as proteolytic cleavage, glycosylation, and disulfide bond formation [1,2,3,4]. Glycosylation is a very common PTM: it is estimated that 50–70% of human proteins are glycosylated [5]. N-glycosylation in yeasts is characterized by hypermannosylation, and terminal α1,3-mannose linkages result in short serum half-life or even immunogenicity of the recombinant glycoproteins, which significantly limits their therapeutic application in humans. Efforts have been made to humanize yeast N-glycosylation, which has been accomplished in P. pastoris [2, 16]
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