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.
Huang
Zhaohua
Z
Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California 94143-0912, USA.
Hwang
Peter
P
Watson
Douglas S
DS
Cao
Limin
L
Szoka
Francis C
FC
D001710:Biotin / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D015394:Molecular Structure; D049329:Nanostructures / Q000737:chemistry*; D009571:Nitrilotriacetic Acid / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D013237:Stereoisomerism; D020349:Surface Plasmon Resonance
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D010446:Peptide Fragments; D006639:Histidine; D001710:Biotin; D009571:Nitrilotriacetic Acid