Protein A
Protein A is a 42 kDa surface protein originally found in the cell wall of the bacteria Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topology, cellular osmolarity, and a two-component system called ArlS-ArlR. It has found use in biochemical research because of its ability to bind immunoglobulins. It is composed of five homologous Ig-binding domains that fold into a three-helix bundle. Each domain is able to bind proteins from many mammalian species, most notably IgGs. It binds the heavy chain within the Fc region of most immunoglobulins and also within the Fab region in the case of the human VH3 family. Through these interactions in serum, where IgG molecules are bound in the wrong orientation (in relation to normal antibody function), the bacteria disrupts opsonization and phagocytosis.[3]
Protein A, Ig-binding domain
SpA
History[edit]
As a by-product of his work on type-specific staphylococcus antigens, Verwey reported in 1940 that a protein fraction prepared from extracts of these bacteria non-specifically precipitated rabbit antisera raised against different staphylococcus types.[4] In 1958, Jensen confirmed Verwey's finding and showed that rabbit pre-immunization sera as well as normal human sera bound to the active component in the staphylococcus extract; he designated this component Antigen A (because it was found in fraction A of the extract) but thought it was a polysaccharide.[5] The misclassification of the protein was the result of faulty tests,[6] but it was not long thereafter (1962) that Löfkvist and Sjöquist corrected the error and confirmed that Antigen A was in fact a surface protein on the bacterial wall of certain strains of S. aureus.[7] The Bergen group from Norway named the protein "Protein A" after the antigen fraction isolated by Jensen.[8]
Other antibody binding proteins[edit]
In addition to protein A, other immunoglobulin-binding bacterial proteins such as protein G, protein A/G and protein L are all commonly used to purify, immobilize or detect immunoglobulins.
Production[edit]
Protein A is produced and purified in industrial fermentation for use in immunology, biological research and industrial applications (see below). Natural (or native) protein A can be cultured in Staphylococcus aureus and contains the five homologous antibody binding regions described above and a C-terminal region for cell wall attachment. Today, protein A is more commonly produced recombinantly in Escherichia coli. (Brevibacillus has also been shown to be an effective host.[15]) Recombinant versions of protein A also contain the five homologous antibody binding domains but may vary in other parts of the structure in order to facilitate coupling to porous substrates.[16] Engineered versions of the protein are also available, the first of which was rProtein A, B4, C-CYS.[17] Engineered versions are multimers (typically tetramers, pentamers or hexamers) of a single domain which has been modified to improve usability in industrial applications.
Research[edit]
Protein A is often coupled to other molecules such as a fluorescent dye, enzymes, biotin, colloidal gold or radioactive iodine without affecting the antibody binding site. Examples including protein A–gold (PAG) stain is used in immunogold labelling, fluorophore coupled protein A for immunofluorescence, and DNA docking strand coupled protein A for DNA-PAINT imaging.[18] It is also widely utilized coupled to magnetic, latex and agarose beads.
Protein A is often immobilized onto a solid support and used as reliable method for purifying total IgG from crude protein mixtures such as serum or ascites fluid, or coupled with one of the above markers to detect the presence of antibodies. The first example of protein A being coupled to a porous bead for purification of IgG was published in 1972.[19] 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.