L-天门冬酰胺酶的可溶性表达研究

Soluble and functional expression of L asparaginase in Escherichia coli

来源于大肠杆菌的L 天门冬酰胺酶是临床化疗的常用药 ,其毒副作用影响了其更为广泛的临床应用。利用蛋白质工程技术对其进行体外分子改造 ,获得毒副作用较小的突变体是克服其毒副作用的有效途径。为此 ,克隆了L 天门冬酰胺酶的前导信号肽、成熟肽编码序列 ,以及其翻译、转录终止序列 ,置于一设计合成的中等强度 β 内酰胺酶启动子调控之下 ,在大肠杆菌中获得可溶性表达 ;表达产物可分泌至细菌周质中 ,并具有催化L 天门冬酰胺解离为天门冬氨酸和氨的反应。本研究为依据酶活性为筛选指标的高通量筛选方法的建立奠定了基础。

L asparaginase from E.coli has been used as anti tumor drug for many years,and its usage in other respects was narrowed by its side effects.Now,It is practical to prepare new mutants without side effects by modern protein biotechnology,such as high thoughput screening of functional mutants from random mutated expression library.In this report,the gene fragment encoding L asparaginase was placed downstream of a moderate β lactamase promotor,and the resulted recombinant bacteria can express and secrete functional L asparaginase to periplasma of the host cell.This study made it possible to establish strategies to screen functional mutants of L asparaginase.