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Application of methyl parathion hydrolase (MPH) as a labeling enzyme
Yang, Wei1,2; Zhou, Ya-Feng1; Dai, He-Ping3; Bi, Li-Jun4; Zhang, Zhi-Ping1; Zhang, Xiao-Hua3; Leng, Yan1; Zhang, Xian-En1; Zhang, XE, Chinese Acad Sci, Wuhan Inst Virol, State Key Lab Virol, Wuhan 430071, Peoples R China
2008-04-01
Source PublicationANALYTICAL AND BIOANALYTICAL CHEMISTRY
ISSN1618-2642
Volume390Issue:8Pages:2133-2140
AbstractMethyl 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 mu L hemolymph from an infected shrimp and differentiated it from a healthy shrimp.; 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 mu L hemolymph from an infected shrimp and differentiated it from a healthy shrimp.
SubtypeArticle
KeywordMethyl Parathion Hydrolase Labeling Enzyme Single-chain Variable Fragment Fusion Protein White Spot Syndrome Virus
Department[Yang, Wei; Zhou, Ya-Feng; Zhang, Zhi-Ping; Leng, Yan; Zhang, Xian-En] Chinese Acad Sci, Wuhan Inst Virol, State Key Lab Virol, Wuhan 430071, Peoples R China; [Yang, Wei] Chinese Acad Sci, Grad Sch, Beijing 100049, Peoples R China; [Dai, He-Ping; Zhang, Xiao-Hua] Chinese Acad Sci, Inst Hydrobiol, Wuhan 430072, Peoples R China; [Bi, Li-Jun] Chinese Acad Sci, Natl Lab Biomacromol, Inst Biophys, Beijing 100101, Peoples R China
Subject AreaAnalytical ; Biochemical Research Methods ; Chemistry
DOI10.1007/s00216-008-1987-y
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine ; Physical Sciences
Indexed BySCI
Language英语
WOS Research AreaBiochemistry & Molecular Biology ; Chemistry
WOS SubjectBiochemical Research Methods ; Chemistry, Analytical
WOS IDWOS:000254755700019
WOS KeywordSPOT SYNDROME VIRUS ; PSEUDOMONAS SP WBC-3 ; IMMUNOASSAY ; ANTIBODY ; SHRIMP ; ELIMINATION ; CONJUGATION ; PROTEINS ; ANTIGENS ; CLONING
Citation statistics
Cited Times:10[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://ir.ihb.ac.cn/handle/152342/8188
Collection期刊论文
Corresponding AuthorZhang, XE, Chinese Acad Sci, Wuhan Inst Virol, State Key Lab Virol, Wuhan 430071, Peoples R China
Affiliation1.Chinese Acad Sci, Wuhan Inst Virol, State Key Lab Virol, Wuhan 430071, Peoples R China
2.Chinese Acad Sci, Grad Sch, Beijing 100049, Peoples R China
3.Chinese Acad Sci, Inst Hydrobiol, Wuhan 430072, Peoples R China
4.Chinese Acad Sci, Natl Lab Biomacromol, Inst Biophys, Beijing 100101, Peoples R China
Recommended Citation
GB/T 7714
Yang, Wei,Zhou, Ya-Feng,Dai, He-Ping,et al. Application of methyl parathion hydrolase (MPH) as a labeling enzyme[J]. ANALYTICAL AND BIOANALYTICAL CHEMISTRY,2008,390(8):2133-2140.
APA Yang, Wei.,Zhou, Ya-Feng.,Dai, He-Ping.,Bi, Li-Jun.,Zhang, Zhi-Ping.,...&Zhang, XE, Chinese Acad Sci, Wuhan Inst Virol, State Key Lab Virol, Wuhan 430071, Peoples R China.(2008).Application of methyl parathion hydrolase (MPH) as a labeling enzyme.ANALYTICAL AND BIOANALYTICAL CHEMISTRY,390(8),2133-2140.
MLA Yang, Wei,et al."Application of methyl parathion hydrolase (MPH) as a labeling enzyme".ANALYTICAL AND BIOANALYTICAL CHEMISTRY 390.8(2008):2133-2140.
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