Cloning, expression, and purification of Pseudomonas aeruginosa keratinase in Escherichia coli AD494(DE3)pLysS expression system.
The DNA encoding keratinase from Pseudomonas aeruginosa was ligated into pET-43b(+) expression vector and transformed into Escherichia coli AD494(DE3)pLysS. After isopropyl beta-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 degrees C, respectively, and was stable at pH 6.0-9.0 and 10-60 degrees C. It was highly inhibited by Cd(2+), Cu(2+), Hg(2+), Ni(2+), Fe(3+), ethylene glycol tetraacetic acid, ethylenediaminetetraacetic acid, and p-chloromercuribenzoate, but activated by Ba(2+), Ca(2+), Mg(2+), Mn(2+), Zn(2+), dithiothreitol, glutathione, and beta-mercaptoethanol. According to substrate specificity results, the purified keratinase was considered to be a metalloprotease.
Department of Food Science, National Taiwan Ocean University, No. 2 Beining Road, Keelung 20224, Taiwan.
Hsin-Hung
HH
Lin
Li-Jung
LJ
Yin
Shann-Tzong
ST
Jiang
D002412:Cations / Q000494:pharmacology; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D010447:Peptide Hydrolases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014169:Transformation, Bacterial
D002412:Cations; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D010447:Peptide Hydrolases; C035855:keratinase