Protein enineering and characterisation of an IgG-binding domain based upon protein G from Streptococcus Group G

Walker, Karen Nicola (1994) Protein enineering and characterisation of an IgG-binding domain based upon protein G from Streptococcus Group G. University of Southampton, Doctoral Thesis.

Abstract

To study the binding and stability of IgG-binding domains of Protein G. a synthetic gene, D-SpG_c2-1, based on the gene encoding the C2 domain of Protein G was designed, synthesised and successfully expressed in pKK223-3. A purification procedure using heat treatment and anion exchange was devised exploiting the high melting temperature and low pI of the C domains. This enabled the purification of the mutated D-SpG_c2-1 protein as well as the wild type. The binding activity of wild type protein was characterised by ELISA and fluorescence spectroscopy. Both methods found the synthetic domain to have a K_d of 300nM. Stopped flow fluorescence studies suggest that a conformational change follows the formation of the initial binary complex. Studies of conformation and conformational stability were carried out using guanidine hydrochloride denaturation, circular dichroism and fluorescence quenching. D-SpG_c2-1 was shown to have a free energy of conformation of 23kJmol^-1. Site-directed mutagenesis was carried out on some of the residues implicated in both the interaction with Fc and in contributing to the hydrophobic core. Surface mutations had little effect on binding or stability, core mutations showed significant reduction in stability. The mutant W48F showed a dramatic loss in affinity for Fc suggesting the involvement of this residue in complex formation. This mutant also caused a 50% reduction in stability without disrupting the structure significantly demonstrating its role in maintaining the hydrophobic core as well as binding.

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