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Studies on the interaction of a single domain of protein G from streptococcus with human Fc and Fab

Studies on the interaction of a single domain of protein G from streptococcus with human Fc and Fab
Studies on the interaction of a single domain of protein G from streptococcus with human Fc and Fab
Protein G (SpG) is a Type III bacterial Fc receptor that binds selectively and non-antigenically to both the Fc and Fab regions of IgG. Here studies are described aimed at understanding the individual contributions to complex stability made by amino acid residues known to make contacts with either the Fab or Fc. Specific amino-acid residues that contribute to these binding interactions via a network of hydrogen bonds have been identified and studied by a programme of site directed mutagenesis. Unique tryptophan residues (W42 or W14) have been used to facilitate equilibrium and pre-equilibrium fluorescence studies to observe binding to Fc or Fab and to determine the Kd for the various equilibria. The effect of secondary substitutions of residues implicated in bond formation to either the Fc or Fab IgG fragment have been determined in order to generate SpG domains with a range of binding affinities for biotechnological uses. SpG species that only bind to Fc or Fab have also been developed. The mutant W42F has been shown by ITC studies to have lost the ability to bind to Fc, and the double mutant T15A-T16A shows no detectable binding to Fab. ITC and kinetic studies at various temperatures have been employed to characterise the thermodynamic parameters underlying binding of wild type or mutated SpG to Fc or Fab. Some restoration of Fc binding activity has been brought about by the placement of a tryptophan at residue 14 (E14W), which may be a useful protein for affinity chromatographic applications as a Kd in the micromolar range is ideal for affinity ligands where easy elution of the bound target protein is required. A dramatic reduction in the affinity of SpG for Fc has been obtained for the E26A mutation, and the N34 residue has been found to be significant in Fc binding, with a 50-fold decrease in affinity for the N34A mutation. The electrostatic nature of the interactions and their sensitivity to changes in pH has also been studied and results showed that Q31E has increased Kd by approximately 6-fold, demonstrating that a negative charge at this position is detrimental to binding. The lower affinity and the influence from the ionisable group would offer advantages for the elution of the Ig from the column.
Muir, Nicola
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Muir, Nicola
34f37c02-d1e4-4457-9b2d-0a74b90d125d
Werner, Jorn
1b02513a-8310-4f4f-adac-dc2a466bd115
Gore, Mike
7bd6db4b-c5a2-4206-8666-b92208ba7979

Muir, Nicola (2009) Studies on the interaction of a single domain of protein G from streptococcus with human Fc and Fab. University of Southampton, School of Biological Sciences, Doctoral Thesis, 306pp.

Record type: Thesis (Doctoral)

Abstract

Protein G (SpG) is a Type III bacterial Fc receptor that binds selectively and non-antigenically to both the Fc and Fab regions of IgG. Here studies are described aimed at understanding the individual contributions to complex stability made by amino acid residues known to make contacts with either the Fab or Fc. Specific amino-acid residues that contribute to these binding interactions via a network of hydrogen bonds have been identified and studied by a programme of site directed mutagenesis. Unique tryptophan residues (W42 or W14) have been used to facilitate equilibrium and pre-equilibrium fluorescence studies to observe binding to Fc or Fab and to determine the Kd for the various equilibria. The effect of secondary substitutions of residues implicated in bond formation to either the Fc or Fab IgG fragment have been determined in order to generate SpG domains with a range of binding affinities for biotechnological uses. SpG species that only bind to Fc or Fab have also been developed. The mutant W42F has been shown by ITC studies to have lost the ability to bind to Fc, and the double mutant T15A-T16A shows no detectable binding to Fab. ITC and kinetic studies at various temperatures have been employed to characterise the thermodynamic parameters underlying binding of wild type or mutated SpG to Fc or Fab. Some restoration of Fc binding activity has been brought about by the placement of a tryptophan at residue 14 (E14W), which may be a useful protein for affinity chromatographic applications as a Kd in the micromolar range is ideal for affinity ligands where easy elution of the bound target protein is required. A dramatic reduction in the affinity of SpG for Fc has been obtained for the E26A mutation, and the N34 residue has been found to be significant in Fc binding, with a 50-fold decrease in affinity for the N34A mutation. The electrostatic nature of the interactions and their sensitivity to changes in pH has also been studied and results showed that Q31E has increased Kd by approximately 6-fold, demonstrating that a negative charge at this position is detrimental to binding. The lower affinity and the influence from the ionisable group would offer advantages for the elution of the Ig from the column.

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Published date: February 2009
Organisations: University of Southampton

Identifiers

Local EPrints ID: 66708
URI: http://eprints.soton.ac.uk/id/eprint/66708
PURE UUID: 68f599ff-f89c-48a7-a4c9-1e02482c531c
ORCID for Jorn Werner: ORCID iD orcid.org/0000-0002-4712-1833

Catalogue record

Date deposited: 14 Jul 2009
Last modified: 14 Mar 2024 02:48

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Contributors

Author: Nicola Muir
Thesis advisor: Jorn Werner ORCID iD
Thesis advisor: Mike Gore

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