Journal
BIOCONJUGATE CHEMISTRY
Volume 20, Issue 8, Pages 1667-1672Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bc900309n
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Funding
- NIH [R01 CA107268, RS10 RR023443, NCRR RR01614]
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Nitrilotriacetic acid (NTA) has moderate affinity (10 mu M) 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.
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