Improved protocol to purify untagged amelogenin - Application to murine amelogenin containing the equivalent P70→T point mutation observed in human amelogenesis imperfecta.
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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DOI：
10.1016/j.pep.2014.09.020
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发表时间：
2015-01
影响因子：
1.6
通讯作者：
Shaw WJ
中科院分区：
文献类型：
--
作者：
Buchko GW;Shaw WJ
釉原蛋白是负责将碳酸化羟基磷灰石转化为牙釉质的主要细胞外蛋白，牙釉质是脊椎动物中最硬和最重矿化的组织。尽管做了很多努力，釉原蛋白调节釉质形成的确切机制还没有完全弄清楚。为了帮助旨在理解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容易的方案是有利的，理想地没有任何标签来帮助亲和纯化。在这里，我们描述了一种改进的方法来纯化毫克量的釉原蛋白，利用其在2%的冰醋酸在低离子强度的条件下的高溶解度。该方法包括在−70 °C下加热冷冻细胞沉淀两次，每次15分钟，其间超声处理2分钟，在2%乙酸（1：250 v/v）中透析两次，然后进行反相色谱。通过在第一步中将冷冻的细胞沉淀物重悬于6 M盐酸胍中，获得产率的进一步提高。乙酸加热法用鼠釉原蛋白说明，该鼠釉原蛋白含有在与釉质形成相关的人釉原蛋白中观察到的相应P70 → T点突变（P71 T），而盐酸胍加热法用野生型鼠釉原蛋白（M180）说明。通过NMR化学位移扰动研究作为蛋白质（0.1-1.8 mM）和NaCl（0-367 mM）浓度的函数来探测P71 T的自组装性质。相对于野生型鼠釉原蛋白的类似研究，P71 T在较低的蛋白质或盐浓度下自缔合，与N-末端附近开始的相互作用。
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 −70 °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 imperfecta (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.