分子伴侣及信号肽对毕赤酵母中分泌蛋白表达水平的影响

目的：探索定位于细胞质、内质网膜及内质网腔中的分子伴侣及其组合对于带有不同信号肽的胞外β-1，3-葡聚糖酶（EXGl）在巴斯德毕赤酵母GS200中表达水平的影响。方法：通过融合PCR技术分别构建带有酵母a交配因子引导肽序列（仅MF）、酵母仅交配因子信号肽序列（ccPre）和重链结合蛋白（Bip）信号肽序列的报告蛋白EXGl的表达质粒pPIC9-EXG1，同时构建分子伴侣基因及其组合的表达质粒pBLArg-IV，然后将2种重组质粒共转化至毕赤酵母宿主菌GS200，转化子经筛选获得共表达菌株，通过测定EXG1酶活来评价分子伴侣与信号肽对其表达水平的影响。结果：细胞质及内质网膜上的分子伴侣Sec61a、Sec61B及胞质中的分子伴侣Ydjl、Ssal、Hsp104及其组合对各种信号肽引导的报告蛋白EXG1的表达水平没有显著影响。然而，内质网腔中的分子伴侣Bip、EroI、PDI与HacI组合能显著提高报告蛋白EXG1的表达水平，其中，以aMF或ctPre作为信号肽引导的报告蛋白EXG1的表达水平分别提高了2．6倍和3．8倍，以Bip信号肽引导的报告蛋白EXGl的表达水平提高了20％～45％，而对于以EXG1自身信号肽引导的报告蛋白EXG1的表达水平没有显著影响。结论：在酵母表达体系中，内质网腔中的分子伴侣是报告蛋白EXG1表达水平的重要影响因素．但分子伴侣对于信号肽的选择性还须进一步证明。

Expression Influence of the Chaperones and the Signal Peptides on the Secretion Protein in Pichia pastoris

Objective： To investigate the secretion expression using the chaperones which located in the cytoplasm, endoplasmic reticulum（ER） membrane and the ER ventric where they influent the expression level of exo-β-1,3-glucanase（EXG1） as a report protein with different signal peptides in Pichia pastoris GS200. Methods： The cloned EXG1 cDNA was inserted into the Escherichia coli-yeast shuttle vector of pP1C9, which contained different secretory forms of EXG1 with the aid of the leader peptide sequence of Saccharomyces cerevisiae a-mate factor（aMF）, a-mate factor pre sequence（aPre）, and heavy chain binding protein（Bip） added by the fuse PCR respectively. Meanwhile, different chaperone cDNA were respectively cloned into the plasmid pBLArg-IX. The two constructed expression plasmids, pPIC9-EXG1 and pBLArg-IV, which contained the EXG1 cDNA or chaperone cDNA, were co-transformed into the GS200 cells of the methylotrophic yeast, P.pastoris. The transformants were screened to obtain co-expression strains by the test and analysis of expressed products of shake-flask culture. The expression level of report protein was evaluated using the determination of the EXG1 enzyme activity. Results： The results exhibited that the chaperones, Sec61a and Sec61β located in the ER membrane, and Ydjl, Ssal and Hspl04 located in the cytoplasm had no obviously influence on expression content of EXG1 with different signals. However, the chaperones, Bip, EroI, and PDI in the ER ventric and their combination with HacI displayed to increase the expression products of EXG1 significantly, which had the expression cassette of aMF-EXG1 and aPre-EXG1 up to 2.6 and 3.8 times as compared with the negative control, and the expression cassette of the Bip-EXGI up to about 20%～45%. Furthermore, there was not influence on the EXG1 that had itself signal peptide. Conclusion： According to the results above, the chaperones in the ER ventric revealed to play a important role in the secretion expression of the recombinant EXG1 in P.pastoris expression system. It still cannotbe confirmed that these chaperones prefer any signal peptide of them.