QUANTITATIVE-EVALUATION OF NEUROTENSIN RECEPTOR PURIFICATION BY IMMOBILIZED METAL AFFINITY-CHROMATOGRAPHY

Immobilized metal affinity chromatography has recently been used for p urification of histidine-tagged membrane proteins in the presence of d etergents with varying success. Strong binding to the metal resin is e ssential for purification when expression levels are low. We have inve stigated the influence of tag length and type of detergent on the puri fication of a neurotensin receptor fusion protein expressed in Escheri chia 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 g els was achieved using the nonionic detergents dodecyl maltoside or Tr iton X-100 (53% pure), or a detergent mixture containing the zwitterio nic detergent holamidopropyl)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 pu rification was substantially improved (72% pure) by extending the hist idine tail to 10 residues because this allowed stringent washes at hig h imidazole concentration to remove nonspecifically bound contaminants . This strategy not only resulted in efficient purification of recepto rs from crude membranes, but also worked particularly well for single- step purification from total cell lysates, resulting in 340-fold purif ication of functional neurotensin receptor. (C) 1997 Academic Press.