Construction of a novel system for cell surface display of heterologous proteins on Pichia pastoris.
A versatile vector was developed for heterologous proteins display on the cell surface of Pichia pastoris using the C-terminal half of alpha-agglutinin from Saccharomyces cerevisiae as a membrane anchor under the control of the alcohol oxidase 1 promoter (pAOX1). Multiple cloning sites and the sequence encoding the Xpress epitope (-Asp-Leu-Tyr-Asp-Asp-Asp-Asp-Lys-) were introduced into the vector for insertion of heterologous genes and selective cleavage of target proteins. Enhanced green fluorescence protein (EGFP) was used as a model protein to check the function of this vector. The expression of EGFP on the P. pastoris surface was confirmed by confocal laser scanning microscopy. Fluorescence microscopy and western blot analysis confirmed that EGFP can be successfully cleaved from the cell surface by treating with enterokinase.
State Key Laboratory of Microbial Technology, Life Science School, Shandong University, Jinan, 250100, P.R. China.
Qingjie
Q
Wang
Lei
L
Li
Min
M
Chen
Qingsheng
Q
Qi
Peng George
PG
Wang
D000429:Alcohol Oxidoreductases / Q000235:genetics; D000595:Amino Acid Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D000072235:Mating Factor; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000235:genetics; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
D010455:Peptides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000072235:Mating Factor; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase