stability of the neurotensin receptor nts1 free in detergent solution and immobilized to affinity resin稳定的神经降压素受体nts1洗涤剂溶液中自由和固定化亲和树脂.pdfVIP

Stability of the Neurotensin Receptor NTS1 Free in Detergent Solution and Immobilized to Affinity Resin Jim F. White, Reinhard Grisshammer* National Institute of Neurological Disorders and Stroke, National Institutes of Health, Department of Health and Human Services, Rockville, Maryland, United States of America Abstract Background: 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. Methods and Findings: 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. Conclusion: Our data point towards two ways by which misfolded NTS1 may be generated: Damage by exposure to buf