TITLE

Application of methyl parathion hydrolase (MPH) as a labeling enzyme

AUTHOR(S)
Wei Yang; Ya-Feng Zhou; He-Ping Dai; Li-Jun Bi; Zhi-Ping Zhang; Xiao-Hua Zhang; Yan Leng; Xian-En Zhang
PUB. DATE
April 2008
SOURCE
Analytical & Bioanalytical Chemistry;Apr2008, Vol. 390 Issue 8, p2133
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
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 μL hemolymph from an infected shrimp and differentiated it from a healthy shrimp. [Figure not available: see fulltext.]
ACCESSION #
31520546

 

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