Ding Y S, Owen S M, Lal R B, Ikeda R A
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400, USA.
J Virol. 1998 Apr;72(4):3383-6. doi: 10.1128/JVI.72.4.3383-3386.1998.
Human T-cell leukemia virus type 1 (HTLV-1) is an oncovirus that is clinically associated with adult T-cell leukemia. We report here the construction of a pET19-based expression clone containing HTLV-1 protease fused to a decahistidine-containing leader peptide. The recombinant protein is efficiently expressed in Escherichia coli, and the fusion protein can be easily purified by affinity chromatography. Active mature protease in yields in excess of 3 mg/liter of culture can then be obtained by a novel two-step refolding and autoprocessing procedure. The purified enzyme exhibited Km and Kcat, values of 0.3 mM and 0.143 sec(-1) at pH 5.3 and was inhibited by pepstatin A.
人类嗜T细胞病毒1型（HTLV-1）是一种与成人T细胞白血病临床相关的致瘤病毒。我们在此报告构建了一个基于pET19的表达克隆，该克隆包含与含十肽组氨酸的前导肽融合的HTLV-1蛋白酶。重组蛋白在大肠杆菌中高效表达，融合蛋白可通过亲和层析轻松纯化。然后，通过一种新颖的两步重折叠和自加工程序，可获得产量超过3毫克/升培养物的活性成熟蛋白酶。纯化后的酶在pH 5.3时的Km和Kcat值分别为0.3 mM和0.143秒（-1），并被胃蛋白酶抑制剂A抑制。
