SPR-based interaction studies with small molecular weight ligands using hAGT fusion proteins

An immobilization procedure for protein on surface plasmon resonance sensor (SPR) chips is described. The target protein, cyclophilin D, is thereby genetically linked to a mutant of the human DNA repair protein O⁶-alkylguanine-DNA-alkyltransferase (hAGT). The procedure includes the immobilization of an alkylguanine derivative on the surface by amine coupling and contact of the surface with a solution of the fusion protein (TCypD-hAGT). TCypD-hAGT could be immobilized using buffer solutions of purified protein or cell extracts. High densities of covalently linked proteins were achieved by either procedure. Binding experiments performed with the ligand cyclosporin A indicate relative binding activities close to 100%. The K_D value (12 nM) and the kinetic rate constants k_on (3 × 10⁵ M⁻¹s⁻¹) and k_off (4 × 10⁻³s⁻¹) are given and compared to values determined for cyclophilin D linked to the surface by amide coupling chemistry. The K_D value is in excellent agreement with the K_D value determined in solution by fluorescence titration.