Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

• Published in: Applied and Environmental Microbiology
• Main Authors: Zhang, Li, Liang, Shuli, Zhou, Xinying, Jin, Zi, Jiang, Fengchun, Han, Shuangyan, Zheng, Suiping, Lin, Ying
• Format: Text
• Language: English
• Published: American Society for Microbiology 2013
• Subjects: Biotechnology; Antarc*; Antarctica
• Online Access: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3754174; http://www.ncbi.nlm.nih.gov/pubmed/23835174; https://doi.org/10.1128/AEM.00824-13

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.