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烟酰胺单核苷酸腺苷转移酶的表达、纯化及活性测定 被引量：3

Expression,purification,and enzymatic activity assay of nicotinamide mononucleotide adenylyltransferase
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摘要 目的获得具有较高纯度和酶活性的烟酰胺单核苷酸腺苷转移酶-1(Nmnat1),为进一步对其功能及调控的研究奠定基础。方法在大肠杆菌中进行Nmnat1表达的条件优化,采用Ni-NTA亲和层析法对目的蛋白进行纯化,并通过酶联荧光法测定酶活性。结果 BL21-CondonPlus (DE3)-RIL为适宜的宿主菌,优化的蛋白表达条件为:在2×YT培养基(37μg/ml氯霉素和75μg/ml卡那霉素)中于28℃、0.5mmol/LIPTG(isopropyl beta-D-thiogalactopyranoside,异丙基-β-D-硫代半乳糖苷)诱导8h。纯化获得的Nmnat1蛋白具有较高的纯度和酶活性。结论适宜的宿主菌对Nmnat1蛋白的高效表达至关重要,进行表达条件优化后可获得大量具有较高纯度和酶活性的目的蛋白。
作者 吕小群 张偌瑜 徐学文 管云枫 缪朝玉
机构地区 第二军医大学药学院药理学教研室
出处 《第二军医大学学报》 CAS CSCD 北大核心 2010年第11期1251-1254,共4页 Academic Journal of Second Military Medical University
基金 国家重点基础研究发展计划课题("973计划" 2009CB521902) 国家"重大新药创制"科技专项(2009ZX09303-002) 上海市优秀学科带人计划(10XD1405300) 上海市血管生物学重点实验室开放课题基金(GXY2009001001)~~
关键词 烟酰胺单核苷酸腺苷转移酶 表达 纯化 酶活性
分类号 R977.3 [医药卫生—药品]
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参考文献16

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同被引文献27

  • 1Penberthy WT. Nicotinamide adenine dinucleotide biology and disease. CurrPharmaceut Design, 2009,15 : 1-2.
  • 2Koubova J. Guarente L. How does calorie restriction work. GeneDev, 2003,17(3) :313-321.
  • 3Hassanain HH, Chon SY, Gupta SL. Differential regulation of human indoleanine 2, 3-dioxygenase gene expression by interferons-gamma and-alpha. Analysis of the regulatory reginon of the gene and identification of an interferon-gamma-inducible DNA-binding factor. J Biol Chem, 1993,268 (7) :5077-5084.
  • 4Yahata N, Y uasa S, Araki T. Nicotinamide mononucleotide adenylyltransferase expression in mitochondrial matrix delays wallerian degeneration. J Neurosci, 2009, 29: 6276-6284.
  • 5Zhang T, Berrocal JG, Frizzell KM, et al. Enzymes in theNAD salvage pathway regulate sirtl activity at target gene promoter. J Biol Chem, 2009,284 : 20408-20417.
  • 6Berger F, Lau C, Ziegler M. Regulation of poly( ADP - ribose) polymerasel activity by the phosphorylation state of the nuclear nad biosynthetic enzyme NMN adenylyltransferase 1. PNAS, 2007,104: 3765-3770.
  • 7Nadia Raffaelli, Leonardo Sorei, Adolfo Amiei, et al. Identification of a novel human nicotinamide mononucleotide adenylyltransferase. Biochemical and Biophysical Research Communications, 2002, 297: 835-840.
  • 8Baffle M, Passarella S, Danese G, et al. Rat liver mitochondria synthesize nicotinamide adenine dinucleotide from nicotinarnide mononucleotide and ATP via a putative matrix nicotinamide mononucleotide adenylyltransferase. Biochem Mol Biol Int, 1996, 38 (2) :297-306.
  • 9Arau jo WL, Nunes-Nesi A, Trenkamp S, et al. Inhibition of 2- oxogiutarate dehydrogenase in potato tuber suggests the enzyme is limiting for respiration and confirms its importance in nitrogen assimilation. Plant Physiol, 2008, 148 (4) : 1782-1796.
  • 10Burgos ES, Schramm VL. Weak coupling of ATP hydrolysis to the chemical equilibrium of human nicotinamide phosphoribosyl- transferase. Biochemistry, 2008,47(42 ) : 11086-11096.

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第二军医大学学报
2010年 第11期

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