Abstract
The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration (epichlorohydrin: 30–60 and 1,4-butanediol-diglycidyl ether: 16–36) resulted in higher adsorption to IgM into immobilized metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM. Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently homogeneous on native polyacrylamide gel electrophoresis with a M r of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective and process-compatible alternative to other types of chromatography.
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References
Chaga, G. S. (2001) Twenty-five years of immobilized metal ion affinity chromatography: past, present and future. J. Biochem. Biophys. Methods 49, 313–334.
Yip, T. T. and Hutchens, T. W. (1994) Immobilized metal ion affinity chromatography. Mol. Biotechnol. 1, 151–164.
Porath J. (1992) Immobilized metal ion affinity chromatography. Protein Expr. Purif. 3, 263–281.
Armisén, P., Mateo, C., Cortés, E., et al. (1999) Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports. J. Chromatography A 848, 61–70.
Pieper, R., Su, Q., Gatlin, C. L., Huang, S.-T., Anderson, N. L., and Steiner, S. (2003) Multi-component immunoaffinity subtraction chromatography: an innovative step towards a comprehensive survey of the human plasma proteome. Proteomics 3, 422–432.
Lee, W.-C. and Lee, K. H. (2004). Applications of affinity chromatography in proteomics. Anal. Biochem. 324, 1–10.
Pavlickova, P., Scneier, E. M., and Hug, H. (2004) Advances in recombinant antibody microarrays. Clin. Chim. Acta 343, 17–35.
Josic, D. and Lim, Y-P. (2001) Analytical and preparative methods for purification of antibodies. Food Technol. Biotechnol. 39, 215–226.
Todorova-Balvay, D., Pitiot, O., Bourhim, M., Srikrishnan, T., and Vijayalajshmi, M. (2004) Immobilized metal-ion affinity chromatography of human antibodies and their proteolytic fragments. J. Chromatogr. B 808, 57–62.
Nisnevitch, M. and Firer, M. A. (2001) The solid phase in affinity chromatography: strategies for antibody attachment. J. Biochem. Biophys. Methods 49, 467–480.
Karmali, A., Pacheco, R., Tata, R., and Brown, P. R. (2001) Substitutions of Thr-103-Ile and Trp-138-Gly in amidase from Pseudomonas aeruginosa are responsible for altered kinetic properties and enzyme instability. Mol. Biotechnol. 17, 201–212.
Brown, P. R. and Clarke, P. H. (1972) amino acid substitution in an amidase produced by an acetanilide-utilizing mutant of Pseudomonas aeruginosa. J. Gen. Microbiol. 70, 287–298.
Martins, S., Andrade, J., Karmali, A., and Serralheiro, M. L. (2005) Characterization of monoclonal antibodies against mutant amidase from Pseudomonas aeruginosa. Mol. Biotechnol. 30, 207–219.
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
Hames, B. D. (1981) An introduction to polyacrylamide gel electrophoresis. In: Gel Electrophoresis of Proteins (Hames, B. D. and Rickwood, D., eds.) IRL Press, pp. 1–86.
Wray, W., Boulikes, T., Wray, V. P., and Hancock, R. (1981) Silver stain of proteins in polyacrylamide gels. Anal. Biochem. 118, 197–202.
Novo, C., Karmali, A., Clemente, A., and Brown, P. R. (2001) A monoclonal antibody specific for Pseudomonas aeruginosa amidase. Hybridoma 20, 273–279.
Johnstone, A. and Thorpe, R. (1987) Immunoassays. In: Immunochemistry in Practice (Johnstone, A. and Thorpe, R., eds.), Chapter 11, Blackwell Scientific Publications, Oxford, pp. 257–260.
Pacheco, V. and Karmali, A. (1998) Chromatographic behaviour of glucose 1- and 2-oxidases from fungal strains on immobilized metal chelates. J. Indust. Microbiol. Biotechnol. 21, 57–64.
Sulkowski, E. (1987) Protein Purification: Micro to Macro (Burgess, R., ed.) New York, UCLA Symposium on Molecular and Cellular Biology, pp. 149–168.
Sulkowski, E. (1985) Purification of proteins by IMAC. Trends Biotechnol. 3, 1–7.
Hale, J. E. and Beidler, D. E. (1994) Purification of humanized murine and murine monoclonal antibodies using immobilized metal affinity chromatography. Anal. Biochem. 222, 29–33.
Todd, R. J., Johnson, D., and Arnold, F. H. (1994) Multiple-site binding interactions in metal-affinity chromatography. I. Equilibrium binding of engineered histidine-containing cytochromes c. J. Chromatography A 662, 13–26.
Arnold, F. H. (1991) Metal-affinity separations: a new dimension in protein processing. Biotechnology 9, 151–156.
Zachariou, M. and Hearn, M. T. (1995) Protein selectivity in immobilized metal affinity chromatography based on the surface accessibility of aspartic and glutamic acid residues. J. Protein Chem. 14, 419–430.
Zachariou, M. and Hearn, M. T. (1996) Applications of immobilized metal ion chelate complexes as pseudocation exchange adsorbents for protein separation. Biochemistry 35, 202–211.
Suen, R. B., Lin S. C., and Hsu, W. H. (2004) Hydroxyapatite-based immobilized metal affinity adsorbents for protein purification. J. Chromatogr. A. 1048, 31–39.
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Martins, S., Karmali, A., Andrade, J. et al. Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa . Mol Biotechnol 33, 103–113 (2006). https://doi.org/10.1385/MB:33:2:103
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DOI: https://doi.org/10.1385/MB:33:2:103