BIACORE ANALYSIS OF HISTIDINE-TAGGED PROTEINS USING A CHELATING NTA SENSOR CHIP

While BIACORE instruments are routinely used for kinetic measurements and for the determination of binding constants, the immobilization of a ligand onto the sensor chip surface has to be individually optimized for every system. We show here that the histidine (His) tag, routinel y used in protein purification and in detection is an ideal tag for im mobilization, despite the intrinsically low affinity between an immobi lized metal ion and the His tag. This is due to strong rebinding effec ts caused by the high surface density of immobilized Ni2+ -nitrilotria cetic acid (NTA) on the chips used here. The immobilization of the lig and can be adjusted to a low level using the same chip, such that mass transport limitation and rebinding of the analyte to the immobilized ligand is minimal, Nine different proteins with different numbers of H is tags were tested for stable binding to the Ni2+ -NTA surface. Most proteins with one His tag dissociate very rapidly from the Ni2+-NTA su rface, and the K-D for the interaction between His tag and Ni2+-NTA wa s estimated to about 10(-6) M at neutral pH. In contrast, two His tags are usually found to be sufficient for stable binding. The kinetics o f the chaperonin system of Escherichia coli GroEL and GroES were analy sed as a model using this system and found to be very similar to those obtained with covalently immobilized ligands. The sensor chip can be reused many times, because of the powerful regeneration methods. The l igand can be freshly immobilized after each cycle, thus eliminating po tential denaturation upon regeneration as a source of error. (C) 1997 Academic Press.