在酿酒酵母中表达的丙型肝炎病毒NS3丝氨酸蛋白酶结构域的纯化与鉴定

Purification and characterization of the NS3 serine protease domain of hepatitis C virus expressed in Saccharomyces cerevisiae.

作者信息

Markland W, Petrillo R A, Fitzgibbon M, Fox T, McCarrick R, McQuaid T, Fulghum J R, Chen W, Fleming M A, Thomson J A, Chambers S P

机构信息

Vertex Pharmaceuticals Inc., Cambridge, MA 02139-4242, USA.

出版信息

J Gen Virol. 1997 Jan;78 ( Pt 1):39-43. doi: 10.1099/0022-1317-78-1-39.

DOI:10.1099/0022-1317-78-1-39

PMID:9010283

Abstract

cDNA encoding the putative core of the hepatitis C virus NS3 serine protease domain (residues 1-181 of NS3; NS3 (181)) was expressed as an N-terminally (His)6-tagged fusion protein in Saccharomyces cerevisiae. NS3 (181) protease activity was found in soluble cell lysates, and the N-terminal metal-chelating domain facilitated the efficient purification of active enzyme, using immobilized metal affinity chromatography. The purified NS3(181), protease activity was characterized by assaying the trans-cleavage of in vitro transcription-translation generated substrates, and subsequently a previously unobserved cleavage site within the NS5A region was identified. The inhibitory effect of known protease inhibitors was also examined. It is hoped that availability of this method for the expression and purification of the NS3(181) protease will facilitate the development of anti-hepatitis C therapies.

摘要

编码丙型肝炎病毒NS3丝氨酸蛋白酶结构域假定核心（NS3的第1-181位氨基酸；NS3(181)）的cDNA在酿酒酵母中作为N端带有(His)6标签的融合蛋白表达。在可溶性细胞裂解物中发现了NS3(181)蛋白酶活性，并且N端金属螯合结构域利用固定化金属亲和层析促进了活性酶的高效纯化。通过检测体外转录-翻译产生的底物的反式切割来表征纯化的NS3(181)蛋白酶活性，随后在NS5A区域内鉴定出一个先前未观察到的切割位点。还检测了已知蛋白酶抑制剂的抑制作用。希望这种用于NS3(181)蛋白酶表达和纯化的方法将有助于抗丙型肝炎疗法的开发。