Site directed mutagenesis of an IgG binding protein based upon Protein A from Staphylococcus aureus
Site directed mutagenesis of an IgG binding protein based upon Protein A from Staphylococcus aureus
Protein A is a cell surface protein of Staphylococcus aureus which contains five immunoglobulin binding domains, each of which is able to bind to the Fc region of IgG. A synthetic gene encoding two IgG binding domains has previously been constructed and expressed as a fusion protein with 81 amino acids of the N-terminal of inactive bovine DNase 1. This protein (81-SpAB*-2 exhibits similar IgG binding characteristics as native Protein A (Popplewell, et al 1991). Chemical modification of the tyrosine residues in 81-SpAB*-2 using tetranitromethane (TNM) results in a reduction in the affinity for IgG similar to that observed when Protein A is modified by the same reagent. The mutation of the sole tyrosine residue (Tyr 18) in each binding domain of 81-SpAB*-2 by substitution with a variety of charged amino acids results in a shift in the pH dependence of Fc binding. Additional amino acid substitutions were accomplished in an attempt to produce a protein that is either sensitive to subtle changes in pH, allowing the protein to dissociate from IgG under less acidic conditions than are presently needed for the separation from native Protein A, or is able to bind human IgG3. Arginine residues were removed from the IgG binding domains and replaced with lysine residues, rendering these domains insensitive to digestion by endoprotease Arg C. This allows the removal of DNase 1 moiety with this endoprotease, cleaving close to the point of fusion. An hydroxylamine sensitive asparagine-glycine bond was also added into the junction between DNase 1 and the IgG binding domains allowing chemical cleavage. The cleaved proteins possess the same IgG binding affinity as the fusion protein and are not precipitated, at pH values below pH 6 as is the fusion problem. Although not directly involved in binding it was found that the addition of the sequence containing the putative third helix in each binding domain (Torigoe, et al 1990a/b) increases the affinity of the protein for IgG implying that this region is important in the stabilisation of tertiary structure.
University of Southampton
1992
Ferrris, William Frank
(1992)
Site directed mutagenesis of an IgG binding protein based upon Protein A from Staphylococcus aureus.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Protein A is a cell surface protein of Staphylococcus aureus which contains five immunoglobulin binding domains, each of which is able to bind to the Fc region of IgG. A synthetic gene encoding two IgG binding domains has previously been constructed and expressed as a fusion protein with 81 amino acids of the N-terminal of inactive bovine DNase 1. This protein (81-SpAB*-2 exhibits similar IgG binding characteristics as native Protein A (Popplewell, et al 1991). Chemical modification of the tyrosine residues in 81-SpAB*-2 using tetranitromethane (TNM) results in a reduction in the affinity for IgG similar to that observed when Protein A is modified by the same reagent. The mutation of the sole tyrosine residue (Tyr 18) in each binding domain of 81-SpAB*-2 by substitution with a variety of charged amino acids results in a shift in the pH dependence of Fc binding. Additional amino acid substitutions were accomplished in an attempt to produce a protein that is either sensitive to subtle changes in pH, allowing the protein to dissociate from IgG under less acidic conditions than are presently needed for the separation from native Protein A, or is able to bind human IgG3. Arginine residues were removed from the IgG binding domains and replaced with lysine residues, rendering these domains insensitive to digestion by endoprotease Arg C. This allows the removal of DNase 1 moiety with this endoprotease, cleaving close to the point of fusion. An hydroxylamine sensitive asparagine-glycine bond was also added into the junction between DNase 1 and the IgG binding domains allowing chemical cleavage. The cleaved proteins possess the same IgG binding affinity as the fusion protein and are not precipitated, at pH values below pH 6 as is the fusion problem. Although not directly involved in binding it was found that the addition of the sequence containing the putative third helix in each binding domain (Torigoe, et al 1990a/b) increases the affinity of the protein for IgG implying that this region is important in the stabilisation of tertiary structure.
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Published date: 1992
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Local EPrints ID: 461743
URI: http://eprints.soton.ac.uk/id/eprint/461743
PURE UUID: 4962c51d-7423-4f97-84a3-c1a5ce7a79df
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Date deposited: 04 Jul 2022 18:53
Last modified: 04 Jul 2022 18:53
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Author:
William Frank Ferrris
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