• DOI:10.1006/ABIO.1997.2326
• Corpus ID: 370716

BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip.

@article{Nieba1997BIACOREAO,
  title={BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip.},
  author={Lars Nieba and S E Nieba-Axmann and Anders V. Persson and Markku D. H{\"a}m{\"a}l{\"a}inen and F Edebratt and Annasara Hansson and Jonas Lidholm and Karin Magnusson and Anneli Karlsson and Andreas Pl{\"u}ckthun},
  journal={Analytical biochemistry},
  year={1997},
  volume={252 2},
  pages={
          217-28
        }
}

• L. Nieba, S. E. Nieba-Axmann, +7 authors A. Plückthun
• Published 15 October 1997
• Biology
• Analytical biochemistry

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, routinely used in protein purification and in detection is an ideal tag for immobilization, despite the intrinsically low affinity between an immobilized metal ion and the His tag. This is due to strong rebinding effects caused by the high surface density of immobilized Ni2+-nitrilotriacetic acid (NTA) on the chips used here. The immobilization of the ligand 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 His tags were tested for stable binding to the Ni2+-NTA surface. Most proteins with one His tag dissociate very rapidly from the Ni2+-NTA surface, and the KD for the interaction between His tag and Ni2+-NTA was 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 of the chaperonin system of Escherichia coli GroEL and GroES were analyzed 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 ligand can be freshly immobilized after each cycle, thus eliminating potential denaturation upon regeneration as a source of error.