Cloning, Expression, and Purification of Pseudomonas aeruginosa Keratinase in Escherichia coli AD494(DE3)pLysS Expression System

Abstract

The DNA encoding keratinase from Pseudomonas aeruginosa was ligated into pET-43b(+) expression vector and transformed into Escherichia coli AD494(DE3)pLysS. After isopropyl β-d-thiogalactopyranoside induction, the soluble recombinant keratinase was expressed in E. coli. The keratinase with a molecular mass of 33 kDa was purified to electrophoretical homogeneity after nickel affinity chromatography. It had an optimal pH and temperature of 8.0 and 50 °C, respectively, and was stable at pH 6.0−9.0 and 10−60 °C. It was highly inhibited by Cd2+, Cu2+, Hg2+, Ni2+, Fe3+, ethylene glycol tetraacetic acid, ethylenediaminetetraacetic acid, and p-chloromercuribenzoate, but activated by Ba2+, Ca2+, Mg2+, Mn2+, Zn2+, dithiothreitol, glutathione, and β-mercaptoethanol. According to substrate specificity results, the purified keratinase was considered to be a metalloprotease.