Synthesis and mutagenesis of an IgG-binding protein based upon protein A of Staphylococcus aureus.
A novel protein able to bind with high affinity to the Fc fragment of IgG from a variety of animals has been produced by a gene synthesis approach. The IgG binding is accomplished by the presence of a single or two consecutive domains based upon domain B from protein A of Staphylococcus aureus. The IgG-binding moiety is fused to a peptide containing 21, 53 or 81 amino acids derived from the N-terminus of bovine DNase I. The latter is present to guide the expression of the protein in Escherichia coli into an inclusion body. This facilitates the high expression and recovery of the IgG-binding domains. The binding activity of this fusion protein is very close to that of the native protein A. Site-directed mutagenesis of the fusion protein and subsequent identification of changed binding interactions is reported.
Popplewell
A G
AG
Department of Biochemistry, School of Biological Sciences, University of Southampton, UK.
Gore
M G
MG
Scawen
M
M
Atkinson
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D001666:Binding Sites, Antibody; D003594:Cytoplasmic Granules; D003850:Deoxyribonuclease I / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000235:genetics* / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics*
D007141:Immunoglobulin Fc Fragments; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D003850:Deoxyribonuclease I