Overexpression, purification, and characterization of the recombinant leucine aminopeptidase II of Bacillus stearothermophilus.
For expression of Bacillus stearothermophilus NCIB 8924 leucine aminopeptidase II (LAP II) in Escherichia coli regulated by a T5 promoter, the gene was amplified by polymerase chain reaction and cloned into expression vector pQE-32 to generate pQE-LAPII. The His(6)-tagged enzyme was overexpressed in IPTG-induced E. coli M15 (pQE-LAPII) as a soluble protein and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 425 U/mg protein with a final yield of 76%. The subunit molecular mass of the purified protein was estimated to be 44.5 kDa by SDS-PAGE. The temperature and pH optima for the purified protein were 60 degrees C and 8.0, respectively. Under optimal condition, the purified enzyme showed a marked preference for Leu- p-nitroanilide, followed by Arg- and Lys-derivatives. The His(6)-tagged enzyme was stimulated by Co(2+) ions, but was strongly inhibited by Cu(2+) and Hg(2+) and by the chelating agents, DTT and EDTA. The EDTA-treated enzyme could be reactivated with Co(2+) ions, indicating that it is a cobalt-dependent exopeptidase. Taking the biochemical characteristics together, we found that the recombinant LAP II exhibits no important differences from those properties described for the native enzyme.
Kuo
Lih-Ying
LY
Department of Biology, Tung-Hai University, 181 Taichung-Kan Road, Taichung, Taiwan.
Hwang
Guang-Yuh
GY
Lai
Yu-Jing
YJ
Yang
Shin-Ling
SL
Lin
Long-Liu
LL
D000813:Anilides / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D003035:Cobalt / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007931:Leucyl Aminopeptidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008628:Mercury / Q000494:pharmacology; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D000813:Anilides; D002413:Cations, Divalent; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D003035:Cobalt; C001536:1-leucine-4-nitroanilide; D003300:Copper; D007931:Leucyl Aminopeptidase; C018021:cobaltous chloride; D008628:Mercury