酵母胱硫醚β-合成酶的表达及酶活鉴定  

Expression and Characterization of Yeast Cystathionine β-Synthase

作　　者：王利群[1] 奚学志[1] 曹利民 顾雅萍[1] 孙培培[1] 

机构地区：[1]常州大学制药与生命科学学院,江苏常州213164 [2]常州英赞美科生物科技有限公司,江苏常州213164

出　　处：《中国医药工业杂志》2013年第7期663-668,共6页Chinese Journal of Pharmaceuticals

基　　金：科技部国际合作资助项目(2011DFR30700)

摘　　要：将重组质粒pET45b-yCBS转入E.coli BL21中,构建高效表达酵母胱硫醚β-合成酶(yeast cystathionineβ-synthase,1)的重组菌。研究诱导时菌体浓度、诱导剂IPTG浓度、诱导时间和温度,以及在培养基中添加不同浓度的山梨醇、葡萄糖、甘油对1表达量的影响。使用Ni2+亲和色谱柱纯化重组蛋白,再经His-Trap脱盐柱脱盐,以茚三酮法检测蛋白活性。结果表明,培养基中添加0.05%的山梨醇,重组菌在37℃培养3 h后,添加终浓度为0.1 mmo/L的IPTG于20℃诱导培养15 h,可溶性1表达量达到110 mg/L;纯化后比活为1 320 u/mg。The recombinant vector pET45b-yCBS was constructed and transformed into E. coli BL21 to overexpress yeast cystathionine β-synthase（1）. The effects of various factors on the protein expression were studied, including strain density, the concentration of the inducer IPTG and inducing temperature and time, as well as the cosubstrates such as sorbitol, glucose and glycerin. The expressed protein was purified by Ni2＋ chelating affinity chromatography and desalted by His-Trap desalting column, and its activity was determined with the ninhydrin reaction. The results indicated that the expression level of 1 is the highest when the recombinant strain was induced by 0.1 mmol/L IPTG for 15 h at 20 ℃ after cultured for 3 h at 37 ℃ in the LB medium supplemented with 0.05% sorbitol. The productivity of the soluble 1 reached 110 mg/L. The specific activity of purified 1 is 1 320 u/mg.

关 键 词：酵母胱硫醚β- 合成酶 表达 活性 

分 类 号：Q814[生物学—生物工程]