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

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	Application of methyl parathion hydrolase (MPH) as a labeling enzyme
	Yang, Wei 1,2; Zhou, Ya-Feng 1; Dai, He-Ping 3; Bi, Li-Jun 4; Zhang, Zhi-Ping 1; Zhang, Xiao-Hua 3; Leng, Yan 1; Zhang, Xian-En 1
刊名	ANALYTICAL AND BIOANALYTICAL CHEMISTRY
	2008-04-01
卷号	390 期号:8 页码:2133-2140
关键词	methyl parathion hydrolase labeling enzyme single-chain variable fragment fusion protein white spot syndrome virus
ISSN号	1618-2642
通讯作者	Zhang, XE, Chinese Acad Sci, Wuhan Inst Virol, State Key Lab Virol, Wuhan 430071, Peoples R China
中文摘要	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.
英文摘要	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.
学科主题	Biochemical Research Methods; Chemistry ; Analytical
WOS标题词	Science & Technology ; Life Sciences & Biomedicine ; Physical Sciences
类目[WOS]	Biochemical Research Methods ; Chemistry, Analytical
研究领域[WOS]	Biochemistry & Molecular Biology ; Chemistry
关键词[WOS]	SPOT SYNDROME VIRUS ; PSEUDOMONAS SP WBC-3 ; IMMUNOASSAY ; ANTIBODY ; SHRIMP ; ELIMINATION ; CONJUGATION ; PROTEINS ; ANTIGENS ; CLONING
收录类别	SCI
语种	英语
WOS记录号	WOS:000254755700019
公开日期	2010-10-13
内容类型	期刊论文
源URL	[http://ir.ihb.ac.cn/handle/152342/8188]
专题	水生生物研究所_中科院水生所知识产出（2009年前）_期刊论文
作者单位	1.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
推荐引用方式 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, Xian-En.(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.