一种自剪切内含肽重组酿酒酵母表达优化研究

自剪切内含肽纯化系统在大肠杆菌中已经得到很好的应用。为了在真核生物中应用该系统,以酿酒酵母为宿主,p HR质粒为表达载体,构建微型内含肽ΔI-CM intein酿酒酵母表达系统。以猪免疫球蛋白Ig G的Fc domain为亲和标签,绿色荧光蛋白gfp为报告蛋白,在酿酒酵母GPD强启动子作用下,表达出融合的Fc-intein-gfp蛋白。检测发现融合序列在起始密码子ATG前加入一段Kozak序列有利于gfp表达。在此基础上,将微型内含肽ΔI-CM intein序列替换成密码子优化的ΔI-CMintein-O序列,显著促进gfp蛋白的表达量,初步实现了ΔI-CM intein融合蛋白的高效表达。为新型自剪切内含肽酿酒酵母表达系统的建立和重组蛋白高效纯化的实现奠定了基础。

Optimization study of the expression of self-cleaving mini-intein fused protein in Saccharomyces cerevisiae

Intein-mediated self-cleaving purification tags have been well used in Escherichia coli expression system. For the purpose to apply the system in eukaryotes, we modified the pHR Saccharomyces cerevisiae expression system established in our lab previously. Mycobacterium tuberculosis RecA mini-intein mutant (?I-CM intein) was cloned and green fluorescent protein gene(gfp)was used as a reporter. A tripartite fusion consisting of porcine IgG Fc domain, ?I-CM intein and gfp was expressed under the yeast GPD promoter. Fluorescent microscope observation and flow cytometry analysis revealed that GFP was produced successfully. A Kozak sequence flanking before the ATG start codon and the codon optimization of ?I-CM intein could improve the GFP expression level significantly,indicating the high expression of ?I-CM intein fusion protein. The results lay the foundation for the construction of the novel self-cleaving mini-intein S. cerevisiae expression system and its application in recombinant proteins recovery efficiently.