Anti-Histidine Antibodies as Tools for Reversible Capturing of His-Tagged Fusion Proteins for Subsequent Binding Analysis
The hexahistidine tag is one of most commonly used fusion tags in affinity purification of recombinantly expressed proteins. Real-time binding analysis using Biacore technology allows in-depth characterization of respective association and dissociation patterns of potential binders. Here we tested four commercially available anti-His antibodies for reversible capturing of His-tagged proteins as a basis for a subsequent interaction analysis with non-His-tagged proteins. Anti-penta-, anti-hexa- and anti-RGS-(His)4 antibodies from different distributors were covalently coupled to Biacore sensor chips. Parallel binding studies of 12 heterogeneously sized RGS-(His)6-tagged (Arg-Gly-Ser-(His)6) proteins revealed that the slowest dissociation rate was obtained when using an anti-RGS-(His)4 antibody. Thus in a sandwich binding assay the anti-RGS-(His)4 antibody can be utilized as an appropriate tool for stable yet reversible capturing of RGS-(His)6-tagged proteins with a non-His-tagged protein.
KeywordsSensor Chip Dissociation Rate Constant Covalent Coupling Antibody Surface Binding Stoichiometry
We thank Dr. U. Bierfreund and Dr. W. Jäger from Biacore (GE Healthcare) for support. We thank Dr. H. Seitz (MPI for Molecular Genetics, Berlin, Germany) for providing RGS-(His)6-tagged proteins. F.W.H.'s group is member of Proteome Binders. This work was supported by EU STREP Affinity Proteome to F.W.H.
- Katsamba PS, Navratilova I, Calderon-Cacia M, Fan L, Thornton K, Zhu M, Bos TV, Forte C, Friend D, Laird-Offringa I, Tavares G, Whatley J, Shi E, Widom A, Lindquist KC, Klakamp S, Drake A, Bohmann D, Roell M, Rose L, Dorocke J, Roth B, Luginbuhl B, Myszka DG (2006) Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users. Anal Biochem 352:208–221PubMedCrossRefGoogle Scholar
- Nelson JD, Brunel FM, Jensen R, Crooks ET, Cardoso RM, Wang M, Hessell A, Wilson IA, Binley JM, Dawson PE, Burton DR, Zwick MB (2007) An affinity-enhanced neutralizing antibody against the membrane-proximal external region of human immunodeficiency virus type 1 gp41 recognizes an epitope between those of 2F5 and 4E10. J Virol 81:4033–4043PubMedCrossRefGoogle Scholar
- Rogers J, Schoepp RJ, Schroder O, Clements TL, Holland TF, Li JQ, Li J, Lewis LM, Dirmeier RP, Frey GJ, Tan X, Wong K, Woodnutt G, Keller M, Reed DS, Kimmel BE, Tozer EC (2008) Rapid discovery and optimization of therapeutic antibodies against emerging infectious diseases. Protein Eng Des Sel 21:495–505PubMedCrossRefGoogle Scholar
- Wassaf D, Kuang G, Kopacz K, Wu QL, Nguyen Q, Toews M, Cosic J, Jacques J, Wiltshire S, Lambert J, Pazmany CC, Hogan S, Ladner RC, Nixon AE, Sexton DJ (2006) High-throughput affinity ranking of antibodies using surface plasmon resonance microarrays. Anal Biochem 351:241–253PubMedCrossRefGoogle Scholar