口蹄疫病毒L蛋白酶在大肠杆菌中的表达及生物活性纯化
Expression in Escherichia coli and purification of biologically active L proteinase of foot-and-mouth disease virus.
作者信息
Piccone M E, Sira S, Zellner M, Grubman M J
机构信息
Plum Island Animal Disease Center, Greenport, NY 11944, USA.
出版信息
Virus Res. 1995 Mar;35(3):263-75. doi: 10.1016/0168-1702(94)00084-p.
Abstract
The foot-and-mouth disease virus (FMDV) Lb gene was cloned into bacterial expression vectors under the control of a T7 RNA polymerase promoter. The Lb protein was expressed in both an in vitro transcription-translation system and in Escherichia coli. In vitro expression of a construct containing the Lb gene fused to a portion of the VP4 and 3D genes demonstrated cis cleavage activity that could be blocked by the thiol protease inhibitor E-64. Lb expressed in E. coli was purified from the soluble fraction by metal chelation chromatography. Purified Lb had trans cleavage activity at the L/P1 junction and cleaved the p220 component of the cap-binding protein complex.
摘要
口蹄疫病毒（FMDV）的Lb基因被克隆到受T7 RNA聚合酶启动子控制的细菌表达载体中。Lb蛋白在体外转录-翻译系统和大肠杆菌中均有表达。包含与部分VP4和3D基因融合的Lb基因的构建体的体外表达显示出顺式切割活性，该活性可被巯基蛋白酶抑制剂E-64阻断。在大肠杆菌中表达的Lb通过金属螯合层析从可溶性部分中纯化出来。纯化的Lb在L/P1连接处具有反式切割活性，并切割帽结合蛋白复合物的p220组分。
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