Abstract
The nonantigenic interaction between a recombinant immunoglobulin G (IgG)-binding protein based on the B domain of Protein A fromStaphylococcus aureus (termed SpA1) and the Fc fragment of rabbit IgG has been investigated. The contribution to binding of four putative hydrogen bond contacts between SpA1 and IgG-Fc were examined by the individual substitution of the residues in SpA1 involved in these interactions by others unable to form hydrogen bonds. It was found that the most important of the hydrogen bonds involved Tyr 18 which, when replaced by Phe, resulted in a twofold decrease in IgG-binding affinity. The residues of SpA1 proposed to make close, mainly hydrophobic, contacts with Fc were replaced by residues with potential electrostatic charge to establish the importance of the hydrophobic interaction in the complex. The IgG-binding affinities of the mutant proteins were compared to the wild-type protein by a competitive enzyme-linked immunosorbant assay. The replacement of individual hydrophobic residues by His generated a number of novel IgG-binding proteins with reduced binding affinity at pH 5.0 but which maintained strong binding affinities at pH 8.0. The elution profile of human IgG1-Fc (Fc fragment of human IgG1) from a column made from an immobilized two-domain mutant protein shows that the complex dissociates at a higher pH relative to that of the non-mutated protein thus offering favorable elution characteristics.
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Goward, C. R., Scawen, M. D., Murphy, J. P., and Atkinson, T. (1993) Molecular evolution of bacterial cell-surface proteins.TIBS 18, 136–140.
Boyle, M. D. P. and Reis, K. J. (1987) Bacterial Fc receptors.Biotechnology 5, 697–703.
Uhlén, M., Lindberg, M. and Philipson, L. (1984) The gene for staphylococcal protein A.Immunol. Today 5, 244–248.
Sjöquist, J., Movitz, J., Johansson, I. B., and Hjelm, H. (1972) Localisation of protein A in the bacteria.Eur. J. Biochem. 30, 190–194.
Langone, J. J. (1982) Protein A fromStaphylococcus aureus and related immunoglobulin receptors produced by streptococci and pneumococci.Adv. Immunol. 32, 157–252.
Hárboe, M. and Fölliny, I. (1974) Recognition of two distinct groups of human IgM and IgA based upon binding to two species of staphylococci.Scand. J. Immunol. 3, 471.
Sasso, E. H., Silvermann, G. J., and Mannik, M. (1991) Human IgM molecules that bind staphylococcal protein A contain VHIII H chains.J. Immunol. 147, 1877–1883.
Ljungberg, U. K., Jansson, B., Niss, U., Nilsson, R., Sandberg, B. E. B., and Nilsson, B. (1993) The interaction of different domains of staphylococcal protein A and human IgG., IgA., IgM, and F(ab)2: separation of affinity from specificity.Mol. Immunol. 30, 1279–1285.
Deisenhofer, J. (1981) Crystallographic refinement and atomic models of human Fc fragment and its complex with fragments B of protein A fromStaphylococcus aureus at 2.9Å and 2.8Å.Biochemistry 20, 2361–2370.
Torigoe, H., Shimada, I., Saito, A., Sato, M., and Arata, Y. (1990). Sequential1H NMR assignments and secondary structures of the B domain of staphylococcal protein A: structural changes between the free B domain in solution and the Fc-bound domain in crystal.Biochemistry 29, 8787–8793.
Gouda, H., Torigoe, H., Saito, A., Sato, M., Arata, Y., and Shimada, I. (1992) Three dimensional solution structure of the B domain of staphylococcal protein A: comparisons of the solution and crystal structures.Biochemistry 31, 9665–9672.
Popplewell, A. G., Gore, M. G., Scawen, M. D. and Atkinson, T. (1990) Synthesis and mutagenesis of an IgG-binding protein based upon protein A ofStaphylococcus aureus.Prot. Eng. 4, 963–970.
Bottomley, S. P., Popplewell, A. G., Scawen, M. D., Wan, T., Sutton, B. J. and Gore, M. G. (1994) The stability and unfolding of an IgG-binding protein based upon the B domain of protein A fromStaphylococcus aureus probed by tryptophan substitution and fluorescence spectroscopy.Prot. Eng. 7, 1463–1470.
Smith, P. K., Kroh, R. I., Hermanson, G. Y., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klent, D. C. (1985) Measurement of protein using bicinchoninic acid.Anal. Biochem. 150, 76–85.
Fersht, A. R. (1987) The hydrogen bond in molecular recognition.TIBS 12, 301–304.
Cedergren, L., Andersson, R., Jansson, B., Uhlén, M., and Nilsson, B. (1993). Mutational analysis of the interaction between staphylococcal protein A and human IgG1.Prot. Eng. 6, 441–448.
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Brown, N.L., Bottomley, S.P., Scawen, M.D. et al. A study of the interactions between an IgG-binding domain based on the B domain of staphylococcal protein a and rabbit IgG. Mol Biotechnol 10, 9–16 (1998). https://doi.org/10.1007/BF02745859
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DOI: https://doi.org/10.1007/BF02745859