Stability of the neurotensin receptor NTS1 free in detergent solution and immobilized to affinity resin.
Stability of the neurotensin receptor NTS1 free in detergent solution and immobilized to affinity resin.
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DOI：
10.1371/journal.pone.0012579
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发表时间：
2010-09-07
期刊：
影响因子：
3.7
通讯作者：
Grisshammer R
中科院分区：
文献类型：
--
作者：
White JF;Grisshammer R
重组膜受体的纯化通常通过使用亲和标签，然后如果需要的话进行另外的层析步骤来实现。第二步可能利用特定的受体特性，如配体结合。然而，多个纯化步骤对蛋白质产率和完整性的影响往往记录不多。我们以前已经报道了一个强大的两步纯化程序的重组大鼠神经降压素受体NTS 1给毫克量的功能性受体蛋白。首先，通过使用Ni-NTA树脂的固定化金属亲和层析富集组氨酸标记的受体。其次，通过使用随后的神经降压素柱除去Ni-NTA柱中的残留污染物，得到纯的NTS 1。虽然神经降压素柱中含有功能性受体蛋白，但我们在神经降压素柱中观察到错误折叠的NTS 1。为了研究错误折叠受体的起源，我们通过放射性配体结合、考马斯染色SDS-凝胶光密度测定和蛋白质含量测定来估计每个纯化步骤中功能性和错误折叠NTS 1的量。首先，我们观察到正确折叠的NTS 1由于暴露于去污剂和各种缓冲液组合物而受到损伤，如通过[3 H]神经降压素结合随时间的损失所看到的。第二，暴露于神经降压素亲和树脂产生额外的错误折叠受体蛋白。我们的数据指向错误折叠的NTS 1可能产生的两种方式：通过暴露于缓冲液组分和通过受体与神经降压素亲和树脂的密切接触而造成的损伤。由于NTS 1在洗涤剂溶液中是稳定的神经降压素，我们推测，聚集受体的发生后，与神经降压素树脂接触的NTS 1跨膜核心周围的洗涤剂带扰动的后果。这两种效应都降低了功能性受体蛋白的产量。
Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram quantities of functional receptor protein. First, histidine-tagged receptors are enriched by immobilized metal affinity chromatography using Ni-NTA resin. Second, remaining contaminants in the Ni-NTA column eluate are removed by use of a subsequent neurotensin column yielding pure NTS1. Whilst the neurotensin column eluate contained functional receptor protein, we observed in the neurotensin column flow-through misfolded NTS1. To investigate the origin of the misfolded receptors, we estimated the amount of functional and misfolded NTS1 at each purification step by radio-ligand binding, densitometry of Coomassie stained SDS-gels, and protein content determination. First, we observed that correctly folded NTS1 suffers damage by exposure to detergent and various buffer compositions as seen by the loss of [3H]neurotensin binding over time. Second, exposure to the neurotensin affinity resin generated additional misfolded receptor protein. Our data point towards two ways by which misfolded NTS1 may be generated: Damage by exposure to buffer components and by close contact of the receptor to the neurotensin affinity resin. Because NTS1 in detergent solution is stabilized by neurotensin, we speculate that the occurrence of aggregated receptor after contact with the neurotensin resin is the consequence of perturbations in the detergent belt surrounding the NTS1 transmembrane core. Both effects reduce the yield of functional receptor protein.
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影响因子：
4.8
作者：
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通讯作者：
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DOI：
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发表时间：
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DOI：
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发表时间：
2002-10-15
影响因子：
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