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

Anal Biochem. 2004 Oct 15;333(2):280-8. doi: 10.1016/j.ab.2004.05.058.


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 O(6)-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 x 10(5)M(-1)s(-1)) and k(off) (4 x 10(-3)s(-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.

MeSH terms

  • Cell Extracts
  • Cyclophilins / metabolism
  • Cyclosporine / metabolism
  • Escherichia coli / genetics
  • Humans
  • Kinetics
  • Ligands
  • Molecular Structure
  • Molecular Weight
  • O(6)-Methylguanine-DNA Methyltransferase / genetics
  • O(6)-Methylguanine-DNA Methyltransferase / metabolism*
  • Peptidyl-Prolyl Isomerase F
  • Protein Binding
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Surface Plasmon Resonance / methods*


  • Cell Extracts
  • Peptidyl-Prolyl Isomerase F
  • Ligands
  • Recombinant Fusion Proteins
  • Cyclosporine
  • O(6)-Methylguanine-DNA Methyltransferase
  • Cyclophilins