大肠杆菌L－天门冬酰胺酶Ⅱ基因的高效表达  

High Expression of the Asparaginase Ⅱ Gene of Escherichia coli

作　　者：刘景晶[1] 李晶[1] 吴梧桐[1] 胡梅清[1] 

机构地区：[1]中国药科大学生物化学教研室,南京铁道医学院生物化学教研室

出　　处：《中国药科大学学报》1996年第11期696-700,共5页Journal of China Pharmaceutical University

摘　　要：用一对与大肠杆菌天门冬酰胺酶Ⅱ基因（ＡｎｓＢ）两侧序列互补的寡核苷酸Ｅ１，Ｅ２作引物，以大肠杆菌野生株ＣＰＵ２１０００９的染色体ＤＮＡ为模板，通过ＰＣＲ扩增得到约１．２ｋｂ的ＤＮＡ片段，将此片段插入ｐＫＫ２３３－２的ｔａｃ启动子下游，转化不同的大肠杆菌宿主菌株，结果表明以ＣＰＵ２１０００９为宿主菌时，转化子的酶表达量最高，重组Ｌ－天门冬酰胺酶占菌体总蛋白４８．３％，发酵液酶活力达４００ＩＵ／ｍｌ；比活为４８ＩＵ／ｍｇ蛋白，ＳＤＳ－ＰＡＧＥ显示纯化后的重组Ｌ－天门冬酰胺酶分子量与天然酶相同，均为１４０ＫＤ。The coding sequence of the L-asparaginase Ⅱ gene was amplified from E.coli CPU 210009 by PCR.The produced 1.2 kb fragment was cloned in pKK 233-2 under the tac promotor,and the resulting plasmid was transformed into 5 strains of E. coli.The results showed that the expression level of the transformant,which was obtained by transforming the new construct into E.coli CPU 210009,was higher than that of the other four. The fermentation experiment of this genetic engineered strain showed that the asparaginase activity of the broth culture was 400 IU/ml at the top of the expression.The specific activity of the cells was 48 IU/mg.The amount of the recombinant asparaginase was 48. 3 percent of total bacterial proteins.The molecular weight of the recombinant asparaginase purified into homogeneous was 140 kD, and was the same as native asparaginase.

关 键 词：药物 大肠杆菌 天门冬酰胺酶 基因表达 

分 类 号：TQ464.8[化学工程—制药化工]