Construction of a novel Pichia pastoris cell-surface display system based on the cell wall protein Pir1.
A novel system based on Pir1 from Saccharomyces cerevisiae was developed for cell-surface display of heterologous proteins in Pichia pastoris with the alpha-factor secretion signal sequence. As a model protein, enhanced green fluorescence protein (EGFP) was fused to the N-terminal of the mature peptide of Pir1 (Pir1-a). The expression of fusion protein EGFP-Pir1-a was irregular throughout the P. pastoris cell surface per detection by confocal laser scanning microscopy. A truncated sequence containing only the internal repetitive sequences of Pir1-a (Pir1-b) was used as a new anchor protein in further study. The fusion protein EGFP-Pir1-b was expressed uniformly on the cell surface. The fluorescence intensity of the whole yeast was measured by spectrofluorometer. Western blot confirmed that the fusion proteins were released from cell walls after mild alkaline treatment. The results indicate that a Pir1-based system can express proteins on the surface of P. pastoris and that the fusion proteins do not affect the manner in which Pir1 attaches to the cell wall. The repetitive sequences of Pir1 are required for cell wall retention, and the C-terminal sequence contributes to the irregular distribution of fusion proteins in P. pastoris.
Wang
Qingjie
Q
Life Science School, Shandong University, Jinan, 250100, People's Republic of China.
Li
Lei
L
Chen
Min
M
Qi
Qingsheng
Q
Wang
Peng George
PG
D015153:Blotting, Western; D002473:Cell Wall / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D017930:Genes, Reporter; D005822:Genetic Vectors; D006023:Glycoproteins; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D000072235:Mating Factor; D018613:Microscopy, Confocal; D010455:Peptides / Q000235:genetics; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism*; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013050:Spectrometry, Fluorescence
D005656:Fungal Proteins; D006023:Glycoproteins; C423367:PIR1 protein, S cerevisiae; D010455:Peptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000072235:Mating Factor