Quantitative evaluation of neurotensin receptor purification by immobilized metal affinity chromatography.
Immobilized metal affinity chromatography has recently been used for purification of histidine-tagged membrane proteins in the presence of detergents with varying success. Strong binding to the metal resin is essential for purification when expression levels are low. We have investigated the influence of tag length and type of detergent on the purification of a neurotensin receptor fusion protein expressed in Escherichia coli at a level of about 0.1% of membrane protein. Receptors with six C-terminal histidine residues did not bind to nickel resin in the presence of the anionic detergent sodium dodecyl sulfate. In contrast, partial purification assessed by densitometry of Coomassie-stained gels was achieved using the nonionic detergents dodecyl maltoside or Triton X-100 (53% pure), or a detergent mixture containing the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (46% pure). Linking a highly charged epitope tag to the histidine tail did not affect the nickel-binding properties of receptors. The level of purification was substantially improved (72% pure) by extending the histidine tail to 10 residues because this allowed stringent washes at high imidazole concentration to remove nonspecifically bound contaminants. This strategy not only resulted in efficient purification of receptors from crude membranes, but also worked particularly well for single-step purification from total cell lysates, resulting in 340-fold purification of functional neurotensin receptor.
Grisshammer
R
R
MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
Tucker
J
J
D002846:Chromatography, Affinity / Q000379:methods*; D003902:Detergents; D000939:Epitopes; D004926:Escherichia coli; D009532:Nickel; D009842:Oligopeptides; D010455:Peptides; D018028:Receptors, Neurotensin / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification
D003902:Detergents; D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; D018028:Receptors, Neurotensin; D011993:Recombinant Fusion Proteins; D009532:Nickel; C085706:FLAG peptide