title: Double-hexahistidine tag with high-affinity binding for protein immobilization, purification, and detection on ni-nitrilotriacetic acid surfaces.
abstract: 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.
authors.affiliation: Protein Technologies Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge CB2 4AT, UK.
authors.forename: Farid
authors.initials: F
authors.lastname: Khan
authors.forename: Mingyue
authors.initials: M
authors.lastname: He
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Taussig
mesh_terms: D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D006639:Histidine / Q000737:chemistry*; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry*; D009942:Organometallic Compounds / Q000737:chemistry*; D040081:Protein Array Analysis / Q000379:methods; D012680:Sensitivity and Specificity; D012685:Sepharose / Q000737:chemistry; D013499:Surface Properties; D013997:Time Factors
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D009942:Organometallic Compounds; D049452:Green Fluorescent Proteins; C088321:nickel nitrilotriacetic acid; D006639:Histidine; D012685:Sepharose; D009571:Nitrilotriacetic Acid