基于外泌蛋白质组的酿酒酵母信号肽元件的分析及鉴定

The secretome-derived in-silico characterization of saccharomyces cerevisiae signal peptide motifs for extracellular recombinant protein production

目的分析鉴定酿酒酵母细胞内源性地促进重组蛋白外泌表达的信号肽元件,并应用于重组蛋白的高效合成。方法利用生物信息学软件SignalP4.1、Phobius、Wolf Psort0.2和ProP1.0对已报道的酿酒酵母外泌蛋白质组所对应的信号肽进行计算分析,确定目标信号肽功能元件;再构建融合表达载体,转化酵母细胞,对获得的阳性克隆在摇瓶培养条件下外泌蛋白的表达进行测定验证。结果通过生物信息学分析,从酿酒酵母外泌蛋白质组中确定了16个含有prepro区的信号肽功能元件,以敲除外切β-(1,3)-葡聚糖酶1(EXG1)基因的酿酒酵母W303-1b/(35)ES为宿主菌,采用酵母α-交配因子的信号肽功能元件作为对照,由EXG1的信号肽功能元件引导外泌的融合蛋白活性是α-交配因子的3倍。结论组合外泌蛋白质组和生物信息学分析的研究策略可以快速准确地分离鉴定促进异源蛋白分泌表达的信号肽功能元件,可应用于生物活性蛋白的高效表达。

Objective To characterize the functional motifs of endogenous signal peptides in Saccharomyces cerevisiae for improving the secretion and production of recombinant protein.Methods Based on the reported yeast secretome,the endogenous and exogenous secretory signal peptides were analyzed in-silico using the software SignalP4.1,Phobius,PSORT II,ProP1.0,and NetNGlyc1.0,respectively.Among the identified functional motifs of signal peptides,the prepro-sequence of exo-1,3-beta-glucanase 1(EXG1)was selected to construct the recombinant expression vector and transformed into yeast cells to verify its capacity for recombinant protein production.The positively screened clones in the flask culture were monitored with regard to the enzymatic activities of secretion proteins.Results Sixteen of candidate signal peptides with pro-region were figured out by in-silico analysis.Then the gene encoding EXG1,a beta-glucosidase,amplified by PCR reactions was cloned into the integrated vector pδGAPg constructed previously in our lab,generating plasmid pδGAPg-EXG1.A second reference expression vector pδGAPg-α-Factor-EXG1 was also constructed by swapping of functional motifs of intrinsic signal peptide with alpha-factor signal peptide.The two expression plasmids were transformed into the yeast W303-1b/?ES with the disruption of EXG1,and the supernatants of engineered strains screened were collected to determine the hydrolytic activities using p-nitrophenyl-β-D-glucopyranoside(pNPG)as a substrate.The results indicated that the secreted extracellular proteins derived by EXG1 signal peptide was three-fold higher than that led by alpha-factor signal peptide.Conclusion The strategy of combining extracellular secretome and in-silico analysis is successfully developed to characterize the functional motifs of signal peptide which can be further employed to produce the biopharmaceuticals.