Abstract
BackgroundPurification 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 FindingsTo 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.ConclusionOur 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.
Highlights
Structural and functional work on G-protein-coupled receptors [1,2] such as the rat neurotensin receptor NTS1 [3] requires a reliable source of high-quality material
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
The Ni-NTA column eluate (NiE) contained two major proteins, NTS1 and a 67 kDa protein identified as glucosamine-fructose-6phosphate aminotransferase by mass spectrometry and N-terminal sequence analysis
Summary
Structural and functional work on G-protein-coupled receptors [1,2] such as the rat neurotensin receptor NTS1 [3] requires a reliable source of high-quality material. The E. coli thioredoxin at the receptor C-terminus was found to improve expression levels [6] and a C-terminal decahistidine tag allowed the efficient capture of the NTS1 fusion protein by immobilized metal affinity chromatography (IMAC) [7]. 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. 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. 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
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