title: A simple and efficient protocol for the production of recombinant cathepsin V and other cysteine cathepsins in soluble form in Escherichia coli.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, SI-1000 Ljubljana, Slovenia. marko.novinec@fkkt.uni-lj.si
authors.forename: Marko
authors.initials: M
authors.lastname: Novinec
authors.forename: Miha
authors.initials: M
authors.lastname: Pavšič
authors.forename: Brigita
authors.initials: B
authors.lastname: Lenarčič
mesh_terms: D002403:Cathepsins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D007218:Industrial Microbiology / Q000191:economics / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
chemical_list: D011994:Recombinant Proteins; D002403:Cathepsins