A study of the interactions between an IgG-binding domain based on the B domain of staphylococcal protein A and rabbit IgG.
The nonantigenic interaction between a recombinant immunoglobulin G (IgG)-binding protein based on the B domain of Protein A from Staphylococcus aureus (termed SpA1) and the Fc fragment of rabbit IgG has been investigated. The contribution to binding of four putative hydrogen bond contacts between SpA1 and IgG-Fc were examined by the individual substitution of the residues in SpA1 involved in these interactions by others unable to form hydrogen bonds. It was found that the most important of the hydrogen bonds involved Tyr 18 which, when replaced by Phe, resulted in a twofold decrease in IgG-binding affinity. The residues of SpA1 proposed to make close, mainly hydrophobic, contacts with Fc were replaced by residues with potential electrostatic charge to establish the importance of the hydrophobic interaction in the complex. The IgG-binding affinities of the mutant proteins were compared to the wild-type protein by a competitive enzyme-linked immunosorbent assay. The replacement of individual hydrophobic residues by His generated a number of novel IgG-binding proteins with reduced binding affinity at pH 5.0 but which maintained strong binding affinities at pH 8.0. The elution profile of human IgG1-Fc (Fc fragment of human IgG1) from a column made from an immobilized two-domain mutant protein shows that the complex dissociates at a higher pH relative to that of the non-mutated protein thus offering favorable elution characteristics.
Division of Biochemistry and Molecular Biology, Institute of Biomolecular Sciences, University of Southampton, UK.
N L
NL
Brown
S P
SP
Bottomley
M D
MD
Scawen
M G
MG
Gore
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D006639:Histidine; D006801:Humans; D006860:Hydrogen Bonding; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017510:Protein Folding; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D006639:Histidine