Application of methyl parathion hydrolase (MPH) as a labeling enzyme.
Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70 +/- 13.19 s(-1), which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody-enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [-(Gly-Ser)(5)-] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 microL hemolymph from an infected shrimp and differentiated it from a healthy shrimp. [figure: see text]
Yang
Wei
W
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China.
Zhou
Ya-Feng
YF
Dai
He-Ping
HP
Bi
Li-Jun
LJ
Zhang
Zhi-Ping
ZP
Zhang
Xiao-Hua
XH
Leng
Yan
Y
Zhang
Xian-En
XE
D000818:Animals; D033364:Decapoda; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D008958:Models, Molecular; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D046848:White spot syndrome virus 1 / Q000737:chemistry*
D010455:Peptides; D011993:Recombinant Fusion Proteins; C495424:methyl parathion hydrolase, Pseudomonas; D010744:Phosphoric Monoester Hydrolases