编码S-腺苷-L-高半胱氨酸水解酶的草菇冷诱导基因Cor3功能验证

Cold-Induced Volvariella volvacea Cor3 Gene Encodes an S-Adenosyl-L-homocysteine Hydrolase

将草菇(Volvariella volvacea)冷诱导基因Cor3cDNA序列经EcoR I酶切克隆到原核表达载体pPROK-1中,转化大肠杆菌(Escherichia coli)JM109,经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达、分离纯化发现该蛋白的分子量约为43kD,等电聚焦电泳结果表明,该蛋白的等电点为6.1,HPLC检测说明合成方向的酶活性为每毫克蛋白每分钟4.5μmol,DNTB与同型半胱氨酸颜色反应测定水解方向的酶活性为每毫克蛋白每分钟0.7μmol,根据上述特性可以确定草菇冷诱导基因Cor3所编码的蛋白是S-腺苷同型半胱氨酸水解酶(SAHH)。

The full-length cDNA sequence of Volvariella volvacea Cor3 gene was digested with EcoRI restriction enzyme,ligated to the expression vector pPROK-1,and the recombinant plasmid was transformed into Escherichia coli JM109.The encoded protein was over-expressed in the transformed bacteria by addition of 0.5 mmol/L IPTG to the culture medium and cell-free extracts were prepared by sonication.DEAE-cellulose ion-exchange,Sephacryl S-200 gel filtration and QAE-Sepharose negative ion exchange chromatography yielded a single protein band on SDS-PAGE with a molecular mass of 43 kDa and an isoelectric point of 6.1.The purified protein was identified as S-adenosyl-L-homocysteine hydrolase with a synthetic activity（measured by HPLC） of 4.5 μmol/min/mg protein,and a hydrolytic activity（measured by titration of the product homocysteine with 5,5＇-dithiobis（2-nitrobenzoic acid） of 0.7 μmol/min/mg.