Novinec Marko, Pavšič Miha, Lenarčič Brigita
Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, SI-1000 Ljubljana, Slovenia.
Protein Expr Purif. 2012 Mar;82(1):1-5. doi: 10.1016/j.pep.2011.11.002. Epub 2011 Nov 11.
Cysteine cathepsins are major players in numerous physiologic and pathologic processes and important drug targets. Several different expression systems have been developed for the production of these enzymes. Here we describe a novel, simple and efficient protocol for the production of recombinant cathepsin V and other cysteine cathepsins. Recombinant procathepsin V was expressed in soluble form in the cytoplasm of Escherichia coli and purified in one step by immobilized nickel ion-affinity chromatography, yielding approximately 0.7 mg procathepsin V per liter bacterial culture. The recombinant proenzyme was then autocatalytically activated in vitro by incubation at pH 4.0 and 30 °C. The yield of proenzyme conversion was over 95% and the mature enzyme exhibited potent activity towards several commonly used synthetic substrates. The same protocol also proved successful in the production of several other cysteine procathepsins, such as cathepsin B, demonstrating that this procedure is widely applicable for the production of recombinant papain-like cysteine peptidases.
半胱氨酸组织蛋白酶是众多生理和病理过程中的主要参与者，也是重要的药物靶点。已经开发了几种不同的表达系统来生产这些酶。在此，我们描述了一种生产重组组织蛋白酶V和其他半胱氨酸组织蛋白酶的新颖、简单且高效的方法。重组组织蛋白酶原V以可溶形式在大肠杆菌细胞质中表达，并通过固定化镍离子亲和层析一步纯化，每升细菌培养物可产生约0.7毫克组织蛋白酶原V。然后，通过在pH 4.0和30°C下孵育，使重组酶原在体外自动催化激活。酶原转化率超过95%，成熟酶对几种常用的合成底物表现出强大的活性。同样的方法在生产其他几种半胱氨酸组织蛋白酶原（如组织蛋白酶B）时也被证明是成功的，这表明该方法广泛适用于生产重组木瓜蛋白酶样半胱氨酸肽酶。
