Specific Binding of Integrin alpha_IIbbeta₃ to RGD Peptide Immobilized on a Nitrilotriacetic Acid Chip: a Surface Plasmon Resonance Study

Y.-J. Lu, F. Zhang, and S.-F. Sui^*

Department of Biological Sciences and Biotechnology, State Key Laboratory of Biomembranes, Tsinghua University, Beijing 100084, P. R. China; fax: (86) 10-62784768; E-mail: suisf@mail.tsinghua.edu.cn

^* To whom correspondence should be addressed.

Received December 4, 2001; Revision received January 16, 2002

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Nitrilotriacetic acid has been routinely used in protein purification for its high affinity for His-tagged protein in the presence of Ni²⁺. Here we reported a type of nitrilotriacetic acid chip (NTA-chip) prepared by transferring NTA-DOGS containing a lipid monolayer to a 50 nm thick gold layer deposited on a glass slide. The surface binding ability of His-tagged protein and regeneration of NTA chip were characterized using a synthetic polypeptide P1 (His-His-His-His-His-His-epsilon-aminohexanoic-Gly-Gly-Arg-Gly-Asp-Ser). The effect of divalent cations on integrin binding affinity for RGD ligand was investigated after P1 had been immobilized onto the sensor chip. The results show that the NTA-chip is a useful tool to immobilize His-tagged protein on the chip surface, and can provide a functional orientation for further investigation. The results also show that removing of Ca²⁺ bound on low affinity sites or adding of Mn²⁺ can increase the binding ability of integrin.

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KEY WORDS: nitrilotriacetic acid, surface plasmon resonance, RGD, integrin alpha_IIbbeta₃, His-tagged peptide

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