Harms E, Wehner A, Jennings M P, Pugh K J, Beacham I R, Röhm K H
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Protein Expr Purif. 1991 Apr-Jun;2(2-3):144-50. doi: 10.1016/1046-5928(91)90063-o.
Isoenzyme II of Escherichia coli L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) is among the few enzymes of major therapeutic importance, being used in the treatment of acute lymphoblastic leukemia. We have constructed several inducible expression systems that overproduce asparaginase II from recombinant plasmids. The most efficient of these systems consists of plasmid pTWE1, a derivative of pT7-7, and an ansB- strain of E. coli, CU1783. These cells produce and secrete amounts of asparaginase II that account for 10-15% of the total cellular protein. Most of the active recombinant enzyme can be released from the periplasmic space by a simple osmotic shock procedure. From the resulting material homogeneous asparaginase II was obtained by a two-step procedure. Overall yields of purified asparaginase were 10-15 mg asparaginase II per liter of E. coli culture. The recombinant enzyme appeared identical to conventionally purified preparations.
大肠杆菌L-天冬酰胺酶（L-天冬酰胺酰胺水解酶，EC 3.5.1.1）的同工酶II是少数具有重要治疗意义的酶之一，用于治疗急性淋巴细胞白血病。我们构建了几种可诱导表达系统，这些系统可从重组质粒中过量产生天冬酰胺酶II。其中最有效的系统由质粒pTWE1（pT7 - 7的衍生物）和大肠杆菌的ansB - 菌株CU1783组成。这些细胞产生并分泌的天冬酰胺酶II量占细胞总蛋白的10 - 15%。大多数活性重组酶可通过简单的渗透休克程序从周质空间释放出来。通过两步法从所得材料中获得了均一的天冬酰胺酶II。纯化的天冬酰胺酶的总产率为每升大肠杆菌培养物10 - 15毫克天冬酰胺酶II。重组酶与传统纯化制剂看起来相同。
