Protein A And Protein G Affinity Fusion Systems

SpA (Fig. 1a) is an immunoglobulin-binding surface receptor found in the Gram-positive bacterium Staphylococcus aureus. SpA has found extensive use in immunological and biotechnological research (2-4,11,27,28). SpA binds to the constant (Fc) part of certain immunoglobulins, but the exact biological significance of this property is not clarified (29). Functional and structural analysis of SpA has revealed the presence of a signal peptide, S, which is processed during secretion, five highly homologous domains (E, D, A, B and C) all capable of binding to IgG (30), followed by a cell wall-attaching structure designated XM (31-35) (Fig. 1a). Here, X represents a charged and repetitive region, postulated to interact with the peptidoglycan cell wall (31), whereas M is a region common for Grampositive cell surface-bound receptors containing an LPXaaTG motif, linked to a C-terminal hydrophobic region ending with a charged tail (32-35). It has been demonstrated that the complete M region is required for cell surface anchoring and that the cell wall sorting is accompanied by proteolytic cleavage at the SpA C-terminus and subsequent covalent linking of the surface receptor to the cell wall (32-35). SpA binds to IgG from most mammalian species, including humans. Of the four subclasses of human IgG, SpA binds to IgG1, IgG2, and IgG4 but shows only weak interaction with IgG3 (36).

Several characteristics of the IgG-binding domains of SpA have made them suitable as fusion partners for the production and purification of recombinant proteins: (1) SpA is proteolytically stable. (2) The structure of the IgG-binding domains, each being a three-helix bundle (37-39), appears to be favorable for independent folding of the fusion partner and the fused product because the N- and C-termini of each IgG-binding domain are solvent exposed (38,40). (3) It has been demonstrated to be feasible to introduce sequences accessible for site-specific cleavage of SpA fusion proteins so as to release the target gene product (41). (4) The high solubility of SpA enables the production

Immunoglobulin binding
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