精氨酸脱亚氨酶的表达、纯化和活性研究

目的：建立精氨酸脱亚氨酶的高效表达菌种和纯化工艺路线。方法：人工合成编码支原体精氨酸脱亚氨酶（arginine deiminase, ADI）的基因，构建pBV220-ADI原核表达载体，转染大肠杆菌DH5α中并诱导表达目的蛋白，离子交换层析和分子筛层析法纯化目标蛋白。采用体外精氨酸降解试验测定纯化产物活性。结果：成功构建了原核表达载体pBV220-ADI，基因侧序正确。转化大肠杆菌DH5α后筛选到高水平表达目的蛋白的菌株，目标蛋白以包含体形式存在于胞浆内，表达水平超过全菌体蛋白的35%。采用盐酸胍溶解包含体、低温条件下稀释和透析的方法进行复性。顺次采用阳离子交换和凝胶过滤层析对复性液进行纯化，最终获得纯度达到95%的活性产物。活性测定表明，纯化的ADI比活性为80IU/mg。结论：成功构建了ADI的高效表达菌种，建立了目标物质的分离纯化方法。

Study on the Expression, Purification and activity of arginine deiminase

Objective To develop a high efficient expression, purification system of recombinant Arginine deiminase(ADI).Methods cDNA fragment encoding for mycoplasma ADI was obtained by artificial synthesis and was cloned into prokaryotic expression vector(pBV220). The recombinant ADI was generated by the transformation of the recombinant vector into the host strain DH5α. Anion exchange and gel filtration chromatography was carried out for purification of the recombinant ADI. The biological activity of final product was detected by the assay of agrinine degradation in vitro. Results A prokaryotic expression plasmid pBV220-ADI was generated successfully, and was identified by DNA sequencing; The recombinant protein was highly expressed in DH5α, the proportion of the recombinant protein is exceeded 35% of the whole protein. The inclusion bodies were solubilized with 6 M guanidine hydrochloride under reducing conditions in order to avoid incorrect disulfide-bond formation of the recombinant ADI molecules. Dilution and dialysis at lower degrees temperature were the optimum renaturation methods. After gel filtration, the purity and specific activity of rADI reached 95% and 80 IU/mg respectively. Conclusions A set of protocols for high efficient rADI expression and purification has been established, which is simple, efficient and applicable.