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首页> 外文期刊>Protein Expression and Purification >Improved protocol to purify untagged amelogenin - Application to murine amelogenin containing the equivalent P70 -> T point mutation observed in human amelogenesis imperfecta
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Improved protocol to purify untagged amelogenin - Application to murine amelogenin containing the equivalent P70 -> T point mutation observed in human amelogenesis imperfecta

机译：改进的方案，用于纯化未标记的牙釉蛋白-在鼠牙釉蛋白中的应用，其在人的牙釉质发育不全中观察到了等同的P70-> T点突变

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摘要

Amelogenin is the predominant extracellular protein responsible for converting carbonated hydroxyapatite into dental enamel, the hardest and most heavily mineralized tissue in vertebrates. Despite much effort, the precise mechanism by which amelogenin regulates enamel formation is not fully understood. To assist efforts aimed at understanding the biochemical mechanism of enamel formation, more facile protocols to purify recombinantly expressed amelogenin, ideally without any tag to assist affinity purification, are advantageous. Here we describe an improved method to purify milligram quantities of amelogenin that exploits its high solubility in 2% glacial acetic acid under conditions of low ionic strength. The method involves heating the frozen cell pellet for two 15 min periods at similar to 70 degrees C with 2 min of sonication in between, dialysis twice in 2% acetic acid (1:250 v/v), and reverse phase chromatography. A further improvement in yield is obtained by resuspending the frozen cell pellet in 6 M guanidine hydrochloride in the first step. The acetic acid heating method is illustrated with a murine amelogenin containing the corresponding P70 -> T point mutation observed in an human amelogenin associated with amelogenesis impeifecta (P71T), while the guanidine hydrochloride heating method is illustrated with wild type murine amelogenin (M180). The self-assembly properties of P71T were probed by NMR chemical shift perturbation studies as a function of protein (0.1-1.8 mM) and NaCl (0-367 mM) concentration. Relative to similar studies with wild type murine amelogenin, P71T self-associates at lower protein or salt concentrations with the interactions initiated near the N-terminus. (C) 2014 Elsevier Inc. All rights reserved.
机译：Amelogenin是主要的细胞外蛋白，负责将碳酸的羟基磷灰石转化为牙釉质，而牙釉质是脊椎动物中最坚硬，矿化度最高的组织。尽管付出了很多努力，但牙釉蛋白调节牙釉质形成的确切机制仍未完全了解。为了协助旨在了解牙釉质形成的生化机制的努力，纯化重组表达的釉原蛋白的更为简便的方案是理想的，理想的是没有任何标签来协助亲和纯化。在这里，我们描述了一种纯化方法，用于纯化毫克量的牙釉蛋白，在低离子强度条件下利用其在2％冰醋酸中的高溶解度。该方法包括在类似于70摄氏度的条件下将冷冻的细胞沉淀物加热两次，每次15分钟，中间进行2分钟的超声处理，然后在2％的乙酸（1：250 v / v）中透析两次，然后进行反相色谱分析。通过在第一步中将冷冻的细胞沉淀物重新悬浮在6 M盐酸胍中，可以进一步提高产量。乙酸加热方法以含有与釉质生成不完善相关的人釉蛋白的P70-> T点突变的鼠牙釉蛋白为例进行说明，而野生型鼠釉蛋白蛋白（M180）以盐酸胍为例进行加热。通过NMR化学位移扰动研究探测了P71T的自组装特性，该蛋白是蛋白质（0.1-1.8 mM）和NaCl（0-367 mM）浓度的函数。相对于野生型鼠类牙釉蛋白的类似研究，P71T在较低的蛋白质或盐浓度下会自相关，并在N端附近开始相互作用。 （C）2014 Elsevier Inc.保留所有权利。

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