Kurata Mitsuhiro, Hirata Maki, Watabe Shoji, Miyake Masashi, Takahashi Susumu Y, Yamamoto Yoshimi
Laboratory of Biochemistry and Radiation Biology, Department of Veterinary Sciences, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan.
Protein Expr Purif. 2003 Nov;32(1):119-25. doi: 10.1016/S1046-5928(03)00222-5.
Cytotoxic T-lymphocyte antigen-2 (CTLA-2) is a novel cysteine proteinase inhibitor. The protein sequence is homologous to the proregion of mouse cathepsin L. Here, we report the expression, purification, and characterization of recombinant CTLA-2 (CTLA-2alpha). CTLA-2alpha was cloned into the pET16b vector and the plasmid was transformed into Escherichia coli strain BL21 (DE3) pLysS. The recombinant CTLA-2alpha was highly expressed and purified by His-Bind affinity chromatography, Factor Xa digestion, and hydrophobic chromatography. Throughout these procedures, 3mg recombinant CTLA-2alpha was obtained from 450 ml of bacterial culture medium. The purified protein exhibited inhibitory activities towards certain cysteine proteinases and was properly refolded, as indicated by circular dichroism spectroscopy. Recombinant CTLA-2alpha fully inhibited Bombyx cysteine proteinase (BCP) (overall Kd (Ki*) = 0.23 nM) and and cathepsin L (overall Kd (Ki*) = 0.38 nM). Inhibition of cathepsin H ( Ki = 86 nM) and papain ( Ki = 560 nM) was much weaker, while inhibition of cathepsin B was negligible ( Ki > 1 microM). Our results indicate that mouse CTLA-2alpha is a selective inhibitor of the cathepsin L-like cysteine proteinases.
细胞毒性T淋巴细胞抗原2（CTLA-2）是一种新型半胱氨酸蛋白酶抑制剂。其蛋白质序列与小鼠组织蛋白酶L的前区同源。在此，我们报告重组CTLA-2（CTLA-2α）的表达、纯化及特性。CTLA-2α被克隆到pET16b载体中，该质粒被转化到大肠杆菌BL21（DE3）pLysS菌株中。重组CTLA-2α通过His-Bind亲和层析、因子Xa消化和疏水层析实现高效表达和纯化。在整个过程中，从450毫升细菌培养基中获得了3毫克重组CTLA-2α。纯化后的蛋白质对某些半胱氨酸蛋白酶表现出抑制活性，并且如圆二色光谱所示，其正确复性。重组CTLA-2α完全抑制家蚕半胱氨酸蛋白酶（BCP）（总体解离常数（Ki*）=0.23纳摩尔）和组织蛋白酶L（总体解离常数（Ki*）=0.38纳摩尔）。对组织蛋白酶H（Ki = 86纳摩尔）和木瓜蛋白酶（Ki = 560纳摩尔）的抑制作用要弱得多，而对组织蛋白酶B的抑制作用可忽略不计（Ki > 1微摩尔）。我们的结果表明，小鼠CTLA-2α是组织蛋白酶L样半胱氨酸蛋白酶的选择性抑制剂。
