[16] Purification of proteins using polyhistidine affinity tags

The expression and subsequent purification of recombinant proteins are widely employed in biochemical studies. A powerful purification method involves the use of peptide affinity tags, which are fused to the protein of interest and used to expedite protein purification via affinity chromatography.1,2 A widely employed method utilizes immobilized metal-affinity chromatography (IMAC) to purify recombinant proteins containing a short affinity tag consisting of polyhistidine residues. IMAC is based on the interactions between a transition metal ion (Co2+, Ni2+, Cu2+, Zn2+) immobilized on a matrix and specific amino acid side chains. Histidine is the amino acid that exhibits the strongest interaction with immobilized metal ion matrices, as electron donor groups on the histidine imidazole ring readily form coordination bonds with the immobilized transition metal. Peptides containing sequences of consecutive histidine residues are efficiently retained on IMAC column matrices. Following washing of the matrix material, peptides containing polyhistidine sequences can be easily eluted by either adjusting the pH of the column buffer or adding free imidazole to the column buffer.3 IMAC is a versatile method that can be utilized to rapidly purify polyhistidine affinity-tagged proteins, resulting in 100-fold enrichments in a single purification step.4 Affinity-tagged protein purities can be achieved at up to 95% purity by IMAC in high yield.5,6 Purification using polyhistidine tags has been carried out successfully using a number of expression systems, including Escherichia coli,7 Saccharomyces cerevisiae,8 mammalian cells,9 and baculovirus-infected insect cells.10 However, this purification method may not be sufficient for tagged proteins expressed at low levels that require significantly greater than 100-fold enrichment or for the preparation of highly homogeneous protein samples. In such cases, either the use of a different affinity tag or the use of the polyhistidine tag in conjunction with additional purification techniques should be considered.