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Chapter 17
Purification of Poly-Histidine-Tagged Proteins
Sinéad T. Loughran and Dermot Walls
Abstract
His-tagging is the most widespread and versatile strategy used to purify recombinant proteins for
biochemical and structural studies. Recombinant DNA methods are first used to engineer the addition
of a short tract of poly-histidine tag (His-tag) to the N terminus or C terminus of a target protein. The
His-tag is then exploited to enable purification of the “tagged” protein by immobilised metal affinity
chromatography (IMAC). Here, we describe efficient procedures for the isolation of highly purified
His-tagged target proteins from an Escherichia coli host using IMAC.
Key words: His-Tag, Recombinant protein expression, IMAC, Protein purification, Poly-histidine,
6×Histidine, Affinity chromatography, Ni-NTA
1. Introduction
Affinity tags are highly efficient tools for purifying recombinant
proteins from crude extracts (see Chapter 9). The use of geneti-
cally engineered affinity tags for improved protein purification has
increased greatly in recent years and affinity tags have become
indispensable tools for structural and functional proteomics
initiatives. The most commonly employed method utilises immo-
bilised metal affinity chromatography (IMAC) to purify recombi-
nant proteins containing a short affinity-tag consisting of
poly-histidine (poly His) residues (1). IMAC is based on the
interaction between a transition metal ion (in this case the Nickel
ion, N
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