Oligohis-tags: mechanisms of binding to Ni2+-NTA surfaces.
Since immobilized metal ion affinity chromatography (IMAC) was first reported, several modifications have been developed. Among them, Ni(2+) immobilized by chelation with nitrilotriacetic acid (NTA) bound to a solid support has become the most common method for the purification of proteins carrying either a C- or N-terminal histidine (His) tag. Despite its broad application in protein purification, only little is known about the binding properties of the His-tag, and therefore almost no thermodynamic and kinetic data are available. In this study, we investigated the binding mechanism of His-tags to Ni(2+)-NTA. Different series of oligohistidines and mixed oligohistidines/oligoalanines were synthesized using automated solid-phase peptide synthesis (SPPS). Binding to Ni(2+)-NTA was analyzed both qualitatively and quantitatively with surface plasmon resonance (SPR) using commercially available NTA sensor chips from Biacore. The hexahistidine tag shows an apparent equilibrium dissociation constant (K(D)) of 14 +/- 1 nM and thus the highest affinity of the peptides synthesized in this study. Furthermore, we could demonstrate that two His separated by either one or four residues are the preferred binding motifs within hexahis tag. Finally, elongation of these referred motifs decreased affinity, probably due to increased entropy costs upon binding.
Knecht
Steven
S
Institute of Molecular Pharmacy, Pharmacenter of the University of Basel, Basel, Switzerland.
Ricklin
Daniel
D
Eberle
Alex N
AN
Ernst
Beat
B
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D006639:Histidine / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D009942:Organometallic Compounds / Q000737:chemistry*; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D013499:Surface Properties; D013696:Temperature
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D009942:Organometallic Compounds; C088321:nickel nitrilotriacetic acid; D006639:Histidine; D009571:Nitrilotriacetic Acid