Binding specificity and affinity analysis of an anti-protective antigen peptide reagent using capillary electrophoresis


Peptide biosensor reagents are emerging as an alternative to typical antibody-based detection methods. Peptides can be rapidly isolated using bacterial display methods for new and emerging biothreats and can be chemically synthesized for rapid, large-scale production. With the emergence of peptide biosensor reagents, there is a growing need to develop methods for characterizing binding interactions. Capillary electrophoresis (CE) is a free-solution separation method that is able to determine target and analyte binding association (Kb) and dissociation constants (Kd). In this study, the Kb, Kd, and peptide specificity of an isolated peptide binding reagent to protective antigen (PA) of Bacillus anthracis were evaluated using capillary electrophoresis at 10 and 20 kV. The relative binding specificity was rapidly assessed by measuring the peptide relative mobility shift at 20 kV at nonequilibrium using bovine serum albumin (BSA), horseradish peroxidase (HRP), and an anti-PA monoclonal antibody (mAb). The αPA peptide was shown to be highly specific for PA, with a Kd = 177 nM measured at 20 kV and Kd = 312 nM measured at 10 kV. These results show that peptides from bacterial display libraries can be rapidly tested for specificity and binding affinity, in solution, for use as a potential biosensor reagent against new and emerging biothreats.

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Kogot, J. , Sarkes, D. , Pennington, J. , Pellegrino, P. and Stratis-Cullum, D. (2014) Binding specificity and affinity analysis of an anti-protective antigen peptide reagent using capillary electrophoresis. Advances in Bioscience and Biotechnology, 5, 40-45. doi: 10.4236/abb.2014.51007.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Zhou, B., Carney, C. and Janda, K.D. (2008) Selection and characterization of human antibodies neutralizing Bacillus anthracis toxin. Bioorganic & Medicinal Chemistry, 16, 1903-1913.
[2] Yang, P., Ma, Y., Lee, A.W.M. and Kennedy, R.T. (2009) Measurement of dissociation rate of biomolecular complexes using CE. Electrophoresis, 30, 457-464.
[3] Sooter, L.J., McMasters, S. and Stratis-Cullum, D.N. (2007) Application of capillary electrophoresis to the development and evaluation of aptamer affinity probes— Art. No. 67590T. In: Cullum, B.M. and Porterfield, D.M., Eds., Smart Biomedical and Physiological Sensor Technology V, SPIE, Bellingham, T7590-T7590.
[4] Nguyen, T., Hilton, J.P. and Lin, Q. (2009) Emerging applications of aptamers to micro- and nanoscale biosensing. Microfluidics and Nanofluidics, 6, 347-362.
[5] Stratis-Cullum, D.N., McMasters, S. and Pellegrino, P.M. (2009) Evaluation of relative aptamer binding to campylobacter jejuni bacteria using affinity probe capillary electrophoresis. Analytical Letters, 42, 2389-2402.
[6] Bossi, A., Castelletti, L., Piletsky, S.A., Turner, A.P.F. and Righetti, P.G. (2003) Towards the development of an integrated capillary electrophoresis optical biosensor. Electrophoresis, 24, 3356-3363.
[7] Liu, R.W., Enstrom, A.M. and Lam, K.S. (2003) Combinatorial peptide library methods for immunobiology research. Experimental Hematology, 31, 11-30.
[8] Boder, E.T. and Wittrup, K.D. (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nature Biotechnology, 15, 553-557.
[9] Daugherty, P.S. (2007) Protein engineering with bacterial display. Current Opinion in Structural Biology, 17, 474-480.
[10] Lee, S.Y., Choi, J.H. and Xu, Z.H. (2003) Microbial cell-surface display. Trends in Biotechnology, 21, 45-52.
[11] Rice, J.J., Schohn, A., Bessette, P.H., Boulware, K.T. and Daugherty, P.S. (2006) Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands. Protein Science, 15, 825-836.
[12] Getz, J.A., Rice, J.J. and Daugherty, P.S. (2011) Protease-resistant peptide ligands from a knottin scaffold library. ACS Chemical Biology, 6, 837-844.
[13] VanAntwerp, J.J. and Wittrup, K.D. (2000) Fine affinity discrimination by yeast surface display and flow cytometry. Biotechnology Progress, 16, 31-37.
[14] Daugherty, P.S., Iverson, B.L. and Georgiou, G. (2000) Flow cytometric screening of cell-based libraries. Journal of Immunological Methods, 243, 211-227.
[15] Kenrick, S.A. and Daugherty, P.S. (2010) Bacterial display enables efficient and quantitative peptide affinity maturation. Protein Engineering Design & Selection, 23, 9-17.
[16] Hall, S.S. and Daugherty, P.S. (2009) Quantitative specificity-based display library screening identifies determinants of antibody-epitope binding specificity. Protein Science, 18, 1926-1934.
[17] Gomes, P. and Andreu, D. (2002) Direct kinetic assay of interactions between small peptides and immobilized antibodies using a surface plasmon resonance biosensor. Journal of Immunological Methods, 259, 217-230.
[18] Katsamba, P.S., Park, S. and Laird-Offringa, I.A. (2002) Kinetic studies of RNA-protein interactions using surface plasmon resonance. Methods, 26, 95-104.
[19] Rich, R.L., Cannon, M.J., Jenkins, J., Pandian, P., Sundaram, S., Magyar, R., Brockman, J., Lambert, J. and Myszka, D.G. (2008) Extracting kinetic rate constants from surface plasmon resonance array systems. Analytical Biochemistry, 373, 112-120.
[20] Peter, J.C., Briand, J.P. and Hoebeke, J. (2003) How biotinylation can interfere with recognition: A surface plasmon resonance study of peptide-antibody interactions. Journal of Immunological Methods, 274, 149-158.
[21] Kogot, J.M., Sarkes, D.A., Val-Addo, I., Pellegrino, P.M. and Stratis-Cullum, D.N. (2012) Increased affinity and solubility of peptides used for direct peptide ELISA on polystyrene surfaces through fusion with a polystyrenebinding peptide tag. BioTechniques, 52, 95-102.
[22] Abdiche, Y., Malashock, D., Pinkerton, A. and Pons, J. (2008) Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet. Analytical Biochemistry, 377, 209-217.
[23] Vadim, O., Ernst, K. and Affinity Capillary Electrophoresis. (2003) Electrokinetic Phenomena, CRC Press, Boca Raton.
[24] Kogot, J.M., Zhang, Y., Moore, S.J., Pagano, P., Stratis-Cullum, D.N., Chang-Yen, D., Turewicz, M., Pellegrino, P.M., de Fusco, A., Soh, H.T. and Stagliano, N.E. (2011) Screening of peptide libraries against protective antigen of Bacillus anthracis in a disposable microfluidic cartridge. PLoS One, 6, E26925.
[25] Rice, J.J. and Daugherty, P.S. (2008) Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides. Protein Engineering Design & Selection, 21, 435-442.
[26] Jiang, C.X. and Armstrong, D.W. (2010) Use of CE for the determination of binding constants. Electrophoresis, 31, 17-27.
[27] Stratis-Cullum, D.N., Kogot, J.M., Sarkes, D.A., ValAddo, I. and Pellegrino, P.M. (2011) Bacterial display peptides for use in biosensing applications. In: L.D. Pramatarova, Ed., On Biomimetics, InTech.
[28] Berezovski, M., Drabovich, A., Krylova, S.M., Musheev, M., Okhonin, V., Petrov, A. and Krylov, S.N. (2005) Nonequilibrium capillary electrophoresis of equilibrium mixtures: A universal tool for development of aptamers. Journal of the American Chemical Society, 127, 3165-3171.
[29] Berezovski, M. and Krylov, S.N. (2002) Nonequilibrium capillary electrophoresis of equilibrium mixtures—A single experiment reveals equilibrium and kinetic parameters of protein-DNA interactions. Journal of the American Chemical Society, 124, 13674-5.
[30] Berezovski, M., Nutiu, R., Li, Y.F. and Krylov, S.N. (2003) Affinity analysis of a protein-aptamer complex using nonequilibrium capillary electrophoresis of equilibrium mixtures. Analytical Chemistry, 75, 1382-1386.
[31] Krylov, S.N. and Berezovski, M. (2003) Non-equilibrium capillary electrophoresis of equilibrium mixtures—Appreciation of kinetics in capillary electrophoresis. Analyst, 128, 571-575.

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