Dual activities of human prolidase.
The cDNA encoding human liver prolidase derived from a healthy adult's liver was cloned into the expression vector pPIC9K of Pichia pastoris to construct the recombination expression vector pPIC9K-P. The pPIC9K-P was digested by restriction enzyme Pme I, and then transformed into P. pastoris GS115 by electroporation. Transformants (the insertion recombinant) were induced by methanol to express the recombination protein. The optimal induction conditions (medium pH, methanol concentration and induction time) of the insertion transformant with the highest enzymatic activity were estimated by orthogonal experimental design L9(3(4)). The SDS-PAGE of the recombinant human prolidase (rh-prolidase) in induction medium showed a molecular weight of 73 kDa. The activities of the rh-prolidase and organophosphoric acid anhydrolases (OPAA) were assayed by colorimetric methods. The recombinant enzyme catalyzed the hydrolysis of organophosphorous compound soman as well as the hydrolysis of dipeptide Gly-Pro. Under the optimal induction conditions, the maximal activities of prolidase and OPAA came to 44.1 and 54.8 nmol/min/mg protein respectively in the medium supernatant. The rh-prolidase purified from the supernatant by ion exchange gradient chromatography (DEAE-Sepharose Fast Flow) and gel filtration chromatography (Sephacryl S-200 High Resolution) showed a single band by SDS-PAGE analysis. The purified rh-prolidase could decompose soman via hydrolytic reaction in vitro.
Wang
S H
SH
Department of Biochemical Pharmacology, Beijing Institute of Pharmacology and Toxicology, Tai-Ping Road 27, Beijing 100850, China. wangshuhao@eyou.com
Zhi
Q W
QW
Sun
M J
MJ
D000328:Adult; D043303:Aryldialkylphosphatase / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D004150:Dipeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008099:Liver / Q000201:enzymology; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012999:Soman / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D012999:Soman; D043303:Aryldialkylphosphatase; D004150:Dipeptidases; C022449:proline dipeptidase