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 ␤-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.

Influence of Cell Surface and Nanomechanical Properties on the Flocculation Ability of Industrial Saccharomyces cerevisiae Strains

“…there is a positive correlation between the elasticity of the cell wall, the surface roughness and thickness of the cell, and its flocculation ability. In particular, these results seem to indicate that a high level of flocculation is achieved when the yeast cells have a rough surface and a soft cell wall because this morphological-mechanical synergy could allow for a higher mobility of GPI-anchored proteins (Verstrepen & Klis 2006;Zhang et al, 2013).…”

Geranyl Butyrate Production by Candida antarctica Lipase B-Displaying Pichia pastoris

“…Geraniol (97 % purity) was purchased from Aladdin Reagents Co. (Shanghai, China), and all other chemicals were also obtained commercially and of analytical grade. The recombinant strain of CALB-displaying P. pastoris GS115/CALB-GCW12 was constructed previously in our laboratory [11]. Yeast extract peptone dextrose medium (YPD), buffered glycerol-complex medium (BMGY), and buffered methanol-complex medium (BMMY) were used to culture P. pastoris [11].…”

Scaling-up the synthesis of myristate glucose ester catalyzed by a CALB-displaying Pichia pastoris whole-cell biocatalyst

“…The pPICZ␣A plasmid was purchased from Invitrogen TM . Zeocin resistance plates, Kanamycin resistance plates, tributyrin agar plates, minimal dextrose medium (MD), yeast extract peptone dextrose medium (YPD), buffered glycerol-complex medium (BMGY), and buffered methanol-complex medium (BMMY) were prepared for use as the yeast culture media [17].…”