Modified His-tag fusion vector for enhanced protein purification by immobilized metal affinity chromatography

Affinity tags are highly efficient tools for purifying recombinant proteins from crude extracts. The use of genetically engineered affinity tags for improved protein purification has increased greatly in recent years and affinity tags have become indispensable tools for structural and functional proteomic initiatives. The most commonly employed method utilizes immobilized metal affinity chromatography (IMAC)1 to purify recombinant proteins containing a short affinity-tag consisting of polyhistidine residues. IMAC is a cost-effective, high-throughput single-step purification procedure and virtually all large structural genomics centers use IMAC as their affinity strategy. Polyhistidine tags are usually placed at either the N or the C terminus of recombinant proteins and optimal placement of the tag is protein specific. The poor capture of some proteins is a major drawback to this system, however, probably because the tag is not surface exposed, and many attempts to utilize this strategy end in failure.