Tris-nitrilotriacetic acids of subnanomolar affinity toward hexahistidine tagged molecules.

Nitrilotriacetic acid (NTA) has moderate affinity (10 µM) for hexahistidine (His6) and is widely used in the purification of His6-tagged proteins. The affinity can be increased significantly (10 nM) through multivalency such as using a tris-NTA. We show that the binding affinity of tris-NTA is dependent on the flexibility and length of the spacer between the mono-NTA and the scaffold: the shorter the spacer, the higher the affinity. A series of biotinylated tris-NTA having different spacers were synthesized and used to prepare tris-NTA sensor chips for surface plasmon resonance measurement of binding affinity. Subnanomolar affinity can be achieved with a short spacer. The new high-affinity tris-NTA enables the formation of stable complexes with hexahistidine containing molecules and provides a convenient method to noncovalently attach proteins to various surfaces.