Purification and characterization of recombinant human liver prolidase expressed in Saccharomyces cerevisiae.
The recombinant human liver prolidase (rh-prolidase, EC 3.4.13.9) from the lysate supernatant of engineering yeast Saccharomyces cerevisiae was purified in two steps employing anion-exchange gradient chromatography (DEAE-Sepharose fast flow) and gel filtration chromatography (Sephacryl S-200 high resolution). The purified recombinant protein furnished a single band with a molecular weight of 56 kD. Intensity scanning of the SDS-PAGE gel revealed that the prolidase accounted for more than 90% of total protein. The optimum pH of the catalytic reaction was 8.0. The enzyme was stimulated by Mn2+, but strongly inhibited by Cu2+ and Zn2+. The rh-prolidase expressed in S. cerevisiae had both dipeptidase and organophosphorus acid anhydrolase activity. It catalyzed the hydrolysis of soman and the dipeptide Gly -Pro. In a detoxification test in vitro, purified rh-prolidase was remarkably efficient at eliminating the toxicity of a lethal dose of soman, with the result that mice survived injection of such a dose.
Wang
Shu-Hao
SH
Department of Biochemical Pharmacology, Beijing Institute of Pharmacology and Toxicology, Tai-Ping Road 27, Beijing, 100850, China.
Zhi
Qing-Wen
QW
Sun
Man-Ji
MJ
D003001:Cloning, Molecular; D004150:Dipeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D008099:Liver / Q000201:enzymology*; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins
D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D004150:Dipeptidases; C022449:proline dipeptidase