title: Screening for glycosylphosphatidylinositol-modified cell wall proteins in Pichia pastoris and their recombinant expression on the cell surface.
abstract: Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface.
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, Guangdong, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.forename: Shuli
authors.initials: S
authors.lastname: Liang
authors.forename: Xinying
authors.initials: X
authors.lastname: Zhou
authors.forename: Zi
authors.initials: Z
authors.lastname: Jin
authors.forename: Fengchun
authors.initials: F
authors.lastname: Jiang
authors.forename: Shuangyan
authors.initials: S
authors.lastname: Han
authors.forename: Suiping
authors.initials: S
authors.lastname: Zheng
authors.forename: Ying
authors.initials: Y
authors.lastname: Lin
mesh_terms: D002087:Butyrates / Q000378:metabolism; D002175:Candida / Q000201:enzymology / Q000235:genetics; D061505:Cell Surface Display Techniques; D002473:Cell Wall / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D016681:Genome, Fungal; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000032:analysis*; D008049:Lipase / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000737:chemistry* / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D002087:Butyrates; D005656:Fungal Proteins; D006023:Glycoproteins; D017261:Glycosylphosphatidylinositols; D011993:Recombinant Fusion Proteins; C033592:4-nitrophenyl butyrate; D008049:Lipase; C431349:lipase B, Candida antarctica