Car9-tag介导的重组ω-转氨酶的高效表达和纯化

High-efficiency expression and purification of recombinantω-transaminase mediated by Car9-tag

对来源于紫色色杆菌的ω-转氨酶基因(Genebank:JC032687.1)进行了生物信息学分析,其理论分子量为51213.42,是一种含有多个磷酸功能位点的亲水性蛋白质;利用p ET-28a(+)为载体在宿主细胞E.coli.Rosetta(DE3)中重组表达了融合有Car9标签(Car9-tag)的ω-转氨酶;考察了诱导条件等对ω-转氨酶表达量的影响,摇瓶培养2 h后加入终浓度为1.0 m M的异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导10 h后可获得高表达量的融合ω-转氨酶,其比酶活可达1 547.16 U·g-1;采用了二氧化硅亲和吸附Car9-tagω-转氨酶对ω-转氨酶进行了分离纯化。

Bioinformatics analysis was performed onω-transaminase(ω-TA)gene(Genebank:JC032687.1)derived from Chromobacterium violaceum.The results showed that theω-transaminase with 51213.42 of theoretical molecular weight was a hydrophilic protein with multiple phosphate functional sites.The Car9-tagω-transaminase was expressed in E.coli.Rosetta(DE3)by pET-28a(+)as vector.The effect of the induction conditions on the level expression ofω-transaminase gene was also investigated.When IPTG with the final concentration of 1.0 mM used as inducer was added after the recombinant bacteria was cultured in Luria-Bertani medium for 2 h.The fusion protein could be expressed efficiently after 10 h of IPTG induction,and the enzyme activity was up to 1547.16 U·g-1.The purification ability of Car9 was investigated.Car9-tagω-transaminase was separated and purified by specific adsorption of Car9 and silica.