| Staphylococcal Protein A, or SPA, is a type I membrane protein covalently linked to the cell wall of most strains of the Gram-positive bacterium Staphylococcus aureus. It has high affinity to IgG from various species, for instance human, rabbit and guinea pig but only weak interaction with bovine and mouse. Protein A interacts with antibodies through two distinct binding events: the ''classical binding site on the Fc portion of human IgG1, IgG2, and IgG4, and the ''alternate binding site found on the Fab portion of human IgG, IgM, IgA, and IgE that contain heavy chains of the VH3 subfamily. The most reported molecular weight of protein A from Staphylococcus aureus is about 42,000. The recombinant Streptococci protein A consists of 299 amino acids and has a predicted molecular mass of 33.8 kDa as estimated by SDS-PAGE. Protein A consists of three regions: S, being the signal sequence that is processed during secretion, five homologous IgG binding domains E, D, A, B and C and a cell-wall anchoring regionXM. The truncated protein lacking region X has a molecular weight of about 31kD. The domains are independently capable to bind to the Fc-part of IgG1, IgG2 and IgG4, but shows only weak interaction with IgG3. In addition, all native protein A domains show comparable Fab binding. The binding site for the Fc part of the IgG molecule has been determined in a study of the B domain. The properties of protein A enables it as a powerful affinity ligand for several immunological and purification applications. High selectivity and good physiochemical stability have made protein A the preferred generic ligand for affinity purification of antibodies and molecules tagged with an antibody Fc-region. Protein A can also be used in various immunochemical assays including Western blotting, immunohistochemistry, and ELISA applications by conjugation with different reporter molecules, such as fluorescent dyes (FITC), enzyme markers (peroxidase, beta-galactosidase, alkaline phosphatase), biotin, and colloidal gold. Immunoprecipitation studies with protein A conjugated to beads are also commonly used to purify proteins or protein complexes indirectly through antibodies against the protein or protein complex of interest. |
