Large-scale preparation and characterization of poly(ADP-ribose) and defined length polymers.
Large-scale preparation and characterization of poly(ADP-ribose) and defined length polymers.
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大规模制备聚（ADP-核糖）和定长聚合物并确定其特性。
DOI：
10.1016/j.ab.2012.06.015
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发表时间：
2012-09-15
影响因子：
2.9
通讯作者：
Mitchison TJ
中科院分区：
文献类型：
--
作者：
Tan ES;Krukenberg KA;Mitchison TJ
聚（adp -核糖）[pADPr]是一种由重复adp -核糖单位组成的结构复杂的大型聚合物。它由NAD+通过聚（adp -核糖）聚合酶[PARPs]生物合成，并通过聚（adp -核糖）糖水解酶降解为adp -核糖。PADPr参与许多细胞过程，并通过共价修饰和与特定蛋白的非共价结合发挥生物学功能。人们对pADPr及其结合蛋白之间非共价相互作用的分子识别和结构活性关系知之甚少，部分原因是缺乏大规模的聚合物和确定长度的单位。我们通过优化酶合成和扩大色谱纯化方法，以PARP1和tankyrase 1为原料制备了数百毫克级的多分散pADPr。我们开发并校准了一种阴离子交换色谱法来分配pADPr的大小，并将其扩大到毫克级纯化定义长度的聚合物。此外，我们提出了一种pADPr分析方法来表征PARPs在不同反应条件下产生的pADPr的多分散性，并发现底物蛋白影响pADPr的大小分布。这些方法将有助于pADPr及其结合蛋白的结构和生化研究。
Poly(ADP-ribose) [pADPr] is a large, structurally complex polymer of repeating ADP-ribose units. It is biosynthesized from NAD+ by poly(ADP-ribose) polymerases [PARPs] and degraded to ADP-ribose by poly(ADP-ribose) glycohydrolase. PADPr is involved in many cellular processes and exerts biological function through covalent modification and non-covalent binding to specific proteins. Very little is known about molecular recognition and structure activity relationships for non-covalent interaction between pADPr and its binding proteins, in part because of lack of access to the polymer on large scale and to units of defined lengths. We prepared polydisperse pADPr from PARP1 and tankyrase 1 at hundreds of milligram scale by optimizing enzymatic synthesis and scaling up chromatographic purification methods. We developed and calibrated an anion exchange chromatography method to assign pADPr size and scaled it up to purify defined length polymers on the milligram scale. Furthermore, we present a pADPr profiling method to characterize the polydispersity of pADPr produced by PARPs under different reaction conditions and find that substrate proteins affect the pADPr size distribution. These methods will facilitate structural and biochemical studies of pADPr and its binding proteins.
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影响因子：
4
作者：
Finch, Kristin E.;Knezevic, Claire E.;Hergenrother, Paul J.
通讯作者：
Hergenrother, Paul J.
影响因子：
64.8
作者：
Ahel, Ivan;Ahel, Dragana;West, Stephen C.
通讯作者：
West, Stephen C.
DOI：
10.1073/pnas.0606526103
发表时间：
2006-11-28
影响因子：
11.1
作者：
Andrabi, Shaida A.;Kim, No Soo;Dawson, Ted M.
通讯作者：
Dawson, Ted M.
DOI：
10.2741/3297
发表时间：
2009-01-01
期刊：
Frontiers in bioscience (Landmark edition)
影响因子：
--
作者：
David KK;Andrabi SA;Dawson TM;Dawson VL
通讯作者：
Dawson VL
影响因子：
14.9
作者：
Altmeyer M;Messner S;Hassa PO;Fey M;Hottiger MO
通讯作者：
Hottiger MO