Protein engineering of protein-A from Staphylococcus aureus
Protein engineering of protein-A from Staphylococcus aureus
A synthetic gene encoding an IgG-binding domain, SpAB*, has been designed and constructed using automated DNA synthesis. This domain represents a truncated version of SpAB, one of five homologous IgG-binding domains present in Protein-A (SpA) of Staphylococcus aureus. Two copies of this synthetic gene have been linked together to form a gene encoding a protein with two SpAB* domains. Both synthetic genes have been used to construct gene fusions encoding proteins with the 81 N-terminal amino acids of the mutant bovine DNase I (Q38E,E39Q) protein, followed by a 12 amino acid spacer region, then the SpAB* domain(s). Using this expression system, both the one-domained fusion protein, termed AP10, and the two-domained protein, AP10-2, have been efficiently expressed within E.coli, and rapidly form insoluble inclusion bodies on induction. A purification system has been developed based upon inclusion body isolation. The IgG-binding activities of AP10 and AP10-2 have been compared to SpA using the technique of ELISA. AP10, with only a single IgG-binding domain, binds IgG only weakly. AP10-2 binds IgG with affinity approaching that of SpA per number of IgG-binding sites, and demonstrates a temperature stability, pH dependence and species specificity of activity similar to that of SpA. The N-terminal DNase I residues do not participate in IgG-binding. Site-directed mutagenesis of the SpAB* domains of AP10-2 has been performed using the technique of oligonucleotide cassette replacement. Residues implicated from crystallographic studies to be important in the binding interaction (Deisenhofer, 1981) have been replaced. Tyr-18 of SpAB*, which forms a H-bondwith leu-432 of Fc, has been substituted by Phe-18 to examine the contribution of the H-bond to complex stability. Other replacements have been made at this position to assess the effects of bulkier or charged amino acid side-chains.
University of Southampton
Popplewell, Andrew George
1991
Popplewell, Andrew George
Popplewell, Andrew George
(1991)
Protein engineering of protein-A from Staphylococcus aureus.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
A synthetic gene encoding an IgG-binding domain, SpAB*, has been designed and constructed using automated DNA synthesis. This domain represents a truncated version of SpAB, one of five homologous IgG-binding domains present in Protein-A (SpA) of Staphylococcus aureus. Two copies of this synthetic gene have been linked together to form a gene encoding a protein with two SpAB* domains. Both synthetic genes have been used to construct gene fusions encoding proteins with the 81 N-terminal amino acids of the mutant bovine DNase I (Q38E,E39Q) protein, followed by a 12 amino acid spacer region, then the SpAB* domain(s). Using this expression system, both the one-domained fusion protein, termed AP10, and the two-domained protein, AP10-2, have been efficiently expressed within E.coli, and rapidly form insoluble inclusion bodies on induction. A purification system has been developed based upon inclusion body isolation. The IgG-binding activities of AP10 and AP10-2 have been compared to SpA using the technique of ELISA. AP10, with only a single IgG-binding domain, binds IgG only weakly. AP10-2 binds IgG with affinity approaching that of SpA per number of IgG-binding sites, and demonstrates a temperature stability, pH dependence and species specificity of activity similar to that of SpA. The N-terminal DNase I residues do not participate in IgG-binding. Site-directed mutagenesis of the SpAB* domains of AP10-2 has been performed using the technique of oligonucleotide cassette replacement. Residues implicated from crystallographic studies to be important in the binding interaction (Deisenhofer, 1981) have been replaced. Tyr-18 of SpAB*, which forms a H-bondwith leu-432 of Fc, has been substituted by Phe-18 to examine the contribution of the H-bond to complex stability. Other replacements have been made at this position to assess the effects of bulkier or charged amino acid side-chains.
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Published date: 1991
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Local EPrints ID: 460426
URI: http://eprints.soton.ac.uk/id/eprint/460426
PURE UUID: 446ff227-9e6f-4783-9794-0bdda98aab85
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Date deposited: 04 Jul 2022 18:22
Last modified: 04 Jul 2022 18:22
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Author:
Andrew George Popplewell
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