|
|
Expression, Purification and Characterization of Dimethylarginine Dimethylaminohydrolase |
ZHAO Zhi-jing1, WANG Xue-zhong2, SHEN Wen-ting1, HU Guang1 |
1. Bioengineering Center, Shao Xing Institute of Technology, College of Engineering, Peking University, Shaoxing 312000, China; 2. Shao Xing Kreatiobio Technology Co. Ltd, Shaoxing 312030, China |
|
|
Abstract Dimethylarginine Dimethylaminohydrolase(DDAH) is one of the essential enzyme in enzymatic cycling detection of ADMA, a well recognized biomarker for early (acute myocardial infarction) AMI prognosis and diagnosis. To obtain DDAH with high enzymatic activity, the DDAH was cloned from the genome of Pseudomonas aeruginosa(Schroeter), and expressed in E. coli BL21 at high level of 100mg/L. Recombinant DDAH was purified to more than 95% purity from supernatant of disrupted cells with HisTrap Fast Flow and further polished by cation-exchange chromatography with Q column. The optimum reaction temperature, pH, specific activity, Km and Vmax of the purified DDAH was 35℃, 6.0, 0.5 U/mg, 3.04 mM and 8.07mM/h, respectively. No obvious activity losing was observed when the DDAH stored at termperature from 10℃ to 40℃ and pH from 5.0 to 9.0. Furthermore, more than 95% enzymatic activity was preserved after the purified DDAH was exposed to temperature of 37℃ for one week. Dimerization of the recombinant enzyme had no obvious effect on the enzymatic activity. The yield of recombinant DDAH could reach to 0.4 gram/L with high density fermentation, thus are well suitable for application in large-scale manufacturing of clinical test kit of ADMA and drug screening for DDAH.
|
Received: 11 October 2012
Published: 25 April 2013
|
|
|
|
[1] Boger R H. Asymmetric dimethylarginine (ADMA): a novel risk marker in cardiovascular medicine and beyond. Ann Med, 2006, 38 (2): 126-136. [2] Boger R H, Maas R, Schulze F, et al. Elevated levels of asymmetric dimethylarginine (ADMA) as a marker of cardiovascular disease and mortality. Clin Chem Lab Med, 2005, 43 (10): 1124-1129. [3] Smith C L, Vallance P. Cardiovascular tests: use & limits of biochemical markers -therapeutic measurements of ADMA involved in cardiovascular disorders. Curr Pharm Des, 2005, 11 (17): 2177-2185. [4] Li N, Worthmann H, Deb M, et al. Nitric oxide (NO) and asymmetric dimethylarginine (ADMA): their pathophysiological role and involvement in intracerebral hemorrhage. Neurol Res, 2011, 33 (5): 541-548. [5] Brunini T M, Moss M B, Siqueira M A, et al. Nitric oxide, malnutrition and chronic renal failure. Cardiovasc Hematol Agents Med Chem, 2007, 5 (2): 155-161. [6] Nijveldt R J, Siroen M P, Teerlink T, et al. Elimination of asymmetric dimethylarginine by the kidney and the liver: a link to the development of multiple organ failure? J Nutr, 2004, 134 (10 Suppl): 2848S-2852S; 2853S. [7] Wadham C, Mangoni A A. Dimethylarginine dimethylaminohydrolase regulation: a novel therapeutic target in cardiovascular disease. Expert Opin Drug Metab Toxicol, 2009, 5 (3): 303-319. [8] Fliser D. Perspectives in renal disease progression: the endothelium as a treatment target in chronic kidney disease. J Nephrol, 2010, 23 (4): 369-376. [9] Korandji C, Zeller M, Guilland J C, et al. Asymmetric dimethylarginine (ADMA) and hyperhomocysteinemia in patients with acute myocardial infarction. Clin Biochem, 2007, 40 (1-2): 66-72. [10] Marra M, Bonfigli A R, Testa R, et al. High-performance liquid chromatographic assay of asymmetric dimethylarginine, symmetric dimethylarginine, and arginine in human plasma by derivatization with naphthalene-2,3-dicarboxaldehyde. Anal Biochem, 2003, 318 (1): 13-17. [11] Schwedhelm E. Quantification of ADMA: analytical approaches. Vasc Med, 2005, 10 Suppl 1:S89-95. [12] Horowitz J D, Heresztyn T. An overview of plasma concentrations of asymmetric dimethylarginine (ADMA) in health and disease and in clinical studies: methodological considerations. J Chromatogr B Analyt Technol Biomed Life Sci, 2007, 851 (1-2): 42-50. [13] Ogawa T, Kimoto M, Sasaoka K. Occurrence of a new enzyme catalyzing the direct conversion of NG,NG-dimethyl-L-arginine to L-citrulline in rats. Biochem Biophys Res Commun, 1987, 148 (2): 671-677. [14] 王学忠. 二甲基精氨酸二甲胺水解酶测定方法及其诊断试剂. 中国, 201010595683,2010. Wang X Z. An assay and reagent for determination of dimethylarginine dimethylaminohydrolase’s activity. China, 201010595683, 2010. [15] Smith C L, Birdsey G M, Anthony S, et al. Dimethylarginine dimethylaminohydrolase activity modulates ADMA levels, VEGF expression, and cell phenotype. Biochem Biophys Res Commun, 2003, 308 (4): 984-989. [16] Hasegawa K, Wakino S, Tanaka T, et al. Dimethylarginine dimethylaminohydrolase 2 increases vascular endothelial growth factor expression through Sp1 transcription factor in endothelial cells. Arterioscler Thromb Vasc Biol, 2006, 26 (7): 1488-1494. [17] Ghebremariam Y T, Erlanson D A, Yamada K, et al. Development of a dimethylarginine dimethylaminohydrolase (DDAH) assay for high-throughput chemical screening. J Biomol Screen, 2012, 17 (5): 651-661. [18] Linsky T W, Fast W. Discovery of structurally-diverse inhibitor scaffolds by high-throughput screening of a fragment library with dimethylarginine dimethylaminohydrolase. Bioorg Med Chem, 2012, 20 (18): 5550-5558. [19] Linsky T, Fast W. A continuous, fluorescent, high-throughput assay for human dimethylarginine dimethylaminohydrolase-1. J Biomol Screen, 2011, 16 (9): 1089-1097. [20] Knipp M, Vasak M. A colorimetric 96-well microtiter plate assay for the determination of enzymatically formed citrulline. Anal Biochem, 2000, 286 (2): 257-264. [21] Santa Maria J, Vallance P, Charles I G, et al. Identification of microbial dimethylarginine dimethylaminohydrolase enzymes. Mol Microbiol, 1999, 33 (6): 1278-1279. [22] Plevin M J, Magalhaes B S, Harris R, et al. Characterization and manipulation of the Pseudomonas aeruginosa dimethylarginine dimethylaminohydrolase monomer-dimer equilibrium. J Mol Biol, 2004, 341 (1): 171-184. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
|
Shared |
|
|
|
|
|
Discussed |
|
|
|
|