Abstract
A protocol is described for the isolation of recombinant polyhistidine-tagged membrane proteins from overexpressing Escherichia coli cells. The gene encoding a target membrane protein is cloned into an expression plasmid and then introduced into E. coli cells for overexpression. Membranes from bacterial cells are isolated and the tagged target membrane protein is solubilized in detergent and subsequently bound to an affinity matrix. Tagged proteins are commonly eluted by an excess of a solute that competes for the binding to the matrix. Alternatively, amino acid sequence-specific proteases can be used to cleave off the affinity purification tag directly on the purification column (i.e., on-column cleavage). This selectively releases the target protein and allows subsequent elution. Importantly, this step represents an additional purification step and can significantly increase the purity of the isolated protein.
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