Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.503206
Title: Studies on the interaction of a single domain of protein G from streptococcus with human Fc and Fab
Author: Muir, Nicola
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2009
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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.
Supervisor: Werner, Jorn ; Gore, Michael Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.503206  DOI: Not available
Keywords: QH301 Biology
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