Construction of a Novel <Emphasis Type="Italic">Pichia pastoris</Emphasis> Cell-Surface Display System Based on the Cell Wall Protein Pir1 |
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Authors: | Qingjie Wang Lei Li Min Chen Qingsheng Qi Peng George Wang |
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Institution: | (1) State Key Laboratory of Microbial Technology, Life Science School, Shandong University, Jinan, 250100, PRC |
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Abstract: | 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. |
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