The site-directed mutagenesis of an IgG-binding protein based upon protein A from Staphylococcal aureus
The site-directed mutagenesis of an IgG-binding protein based upon protein A from Staphylococcal aureus
Protein A from Staphylococcal aureus binds with high affinity to the Fc region of IgG. Protein A is composed of 5 highly homologous IgG-binding domains (termed E, D, A, B and C) and a cell wall anchoring region. A synthetic gene, SpAB*, coding for domain B of protein A has been efficiently expressed and purified. Two copies of SpAB* can be easily linked together to form SpAB*-2 which has been shown by ELISA to bind to IgG with similar affinity and specificity to protein A (Popplewell et al., 1991).
In solution domain B is composed of three α helices where helices 2 and 3 interact intimately (Gouda et al., 1992), this tri-helical structure remains intact upon interaction with Fc. SpA_B^*, however, lacks 11 amino acid residues at the C-terminus and therefore is unable to form the third helical motif. Using cassette replacement these additional amino acid residues were inserted into SpA_B^* for form C-SpA_B^* which contains all the amino acid residues necessary to form all three helices. C-SpA_B^* was shown by ELISA to improve the binding affinity 2-3 fold compared to SpA_B^*. C-SpA_B^* and SpA_B^* are devoid of trp residues. A number of individual amino acid residues within both proteins were replaced with trp residues and the fluorescence properties used to study the unfolding mechanism and measure the conformational stability of the proteins.
The presence of the third helical motif was found to increase the conformational stability of the proteins by approximately 0.5kcal/mol. Using the mutants Y18W and 120W the conformational stability of helix 1 was found to be lower than that of helix 2 or 3 determined by use of mutants F34W, I35W, L48W and L55W respectively. This suggests that helix 1 has fewer stabilising interactions than helix 2 or 3. Helices 2 and 3 unfold at higher concentrations of denaturant than helix 1 providing further evidence that these two helices interact together.
University of Southampton
1994
Bottomley, Stephen Paul
(1994)
The site-directed mutagenesis of an IgG-binding protein based upon protein A from Staphylococcal aureus.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Protein A from Staphylococcal aureus binds with high affinity to the Fc region of IgG. Protein A is composed of 5 highly homologous IgG-binding domains (termed E, D, A, B and C) and a cell wall anchoring region. A synthetic gene, SpAB*, coding for domain B of protein A has been efficiently expressed and purified. Two copies of SpAB* can be easily linked together to form SpAB*-2 which has been shown by ELISA to bind to IgG with similar affinity and specificity to protein A (Popplewell et al., 1991).
In solution domain B is composed of three α helices where helices 2 and 3 interact intimately (Gouda et al., 1992), this tri-helical structure remains intact upon interaction with Fc. SpA_B^*, however, lacks 11 amino acid residues at the C-terminus and therefore is unable to form the third helical motif. Using cassette replacement these additional amino acid residues were inserted into SpA_B^* for form C-SpA_B^* which contains all the amino acid residues necessary to form all three helices. C-SpA_B^* was shown by ELISA to improve the binding affinity 2-3 fold compared to SpA_B^*. C-SpA_B^* and SpA_B^* are devoid of trp residues. A number of individual amino acid residues within both proteins were replaced with trp residues and the fluorescence properties used to study the unfolding mechanism and measure the conformational stability of the proteins.
The presence of the third helical motif was found to increase the conformational stability of the proteins by approximately 0.5kcal/mol. Using the mutants Y18W and 120W the conformational stability of helix 1 was found to be lower than that of helix 2 or 3 determined by use of mutants F34W, I35W, L48W and L55W respectively. This suggests that helix 1 has fewer stabilising interactions than helix 2 or 3. Helices 2 and 3 unfold at higher concentrations of denaturant than helix 1 providing further evidence that these two helices interact together.
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Published date: 1994
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Local EPrints ID: 462809
URI: http://eprints.soton.ac.uk/id/eprint/462809
PURE UUID: 6e225c29-2990-4e6a-9559-7bd58a9e5749
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Date deposited: 04 Jul 2022 20:05
Last modified: 04 Jul 2022 20:05
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Author:
Stephen Paul Bottomley
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