Alkaline phosphatase-Strep tag fusion protein binding to streptavidin: resonant mirror studies

The properties of a fusion protein comprising a streptavidin recognition sequence (Strep tag) fused to the C terminus of Escherichia coli alkaline phosphatase are described. The catalytic properties were determined with p-nitrophenyl phosphate and compared to those of the native E. coli alkaline phosphatase. It was found that the Km values were similar in both cases (8 microM for transferase and 2 microM for hydrolase activities) whilst the Vmax values were lower for the fusion protein, possibly due to the presence of misfolded forms. An optical biosensor based on the resonant mirror was used to determine the binding kinetics between the fusion protein and the immobilised streptavidin. The association and dissociation rate constants were determined to be 2.1(+/-0.3) x 10(-2) microM(-1) s(-1) and 11(+/-0.2) x 10(-3) s(-1), respectively, which results in an equilibrium dissociation constant of 0.5 microM. This is larger than previously reported affinities based on titration calorimetry and may be a consequence of the presence of two streptavidin binding sequences on the dimeric alkaline phosphatase simultaneously binding to two subunits of streptavidin.