Double-Hexahistidine Tag with High-Affinity Binding for Protein Immobilization, Purification, and Detection on Ni−Nitrilotriacetic Acid Surfaces

There is a particular need in protein analysis and purification for specific, functional, and generic methods of protein immobilization on solid supports. Here we describe a double-hexahistidine (His6) tag sequence, comprising two hexahistidines separated by an 11-amino acid spacer, which shows at least 1 order of magnitude stronger binding to Ni−NTA-modified surfaces than a conventional single-His6 tag or two single-His6 tags at N- and C-termini. Using, as a model, tagged versions of green fluorescent protein (GFP), stable and tight binding of the double-His6 tag/Ni−NTA interaction was demonstrated by competitive elution from Ni−NTA agarose beads, surface plasmon resonance on a Ni−NTA chip, and ELISA in Ni−NTA microwell plates. Protein purification by Ni−NTA chromatography was improved by a 6−8-fold increase in imidazole concentration required for elution, while the dissociation rate of double-His6 GFP from Ni−NTA chips in SPR (BIAcore) was 10 times slower than for single-His6-tagged proteins. ELISA assays and protein microarrays constructed with double-His6 GFP demonstrated greater detection sensitivity with anti-His antibodies and Ni−NTA conjugates. Moreover, the double-His6 tag could serve simultaneously both for protein immobilization and for detection on surfaces. The double-His6 peptide has the potential to be a universal tag for protein immobilization and detection on arrays and single-step purification of proteins from crude mixtures.