Protein LA, a novel hybrid protein with unique single-chain Fv antibody- and Fab-binding properties.
Existing Ig-binding proteins all suffer from limitations in their binding spectrum. In the pursuit of the ultimate, non-restricted, Ig-binding protein, we have constructed the hybrid protein LA, by fusing four of the Ig kappa light-chain-binding domains of peptostreptococcal protein L with four of the IgGFc- and Fab-binding regions of staphylococcal protein A. Ligand-blot experiments demonstrated that the L and the A components were both functional in the hybrid, as the protein was shown to bind purified kappa light chains and IgGFc. Protein LA bound human Ig of different classes and IgG from a wide range of mammalian species. IgG, IgM and IgA were purified from human serum and saliva by affinity chromatography on protein LA agarose. Similarly, single-chain Fv (scFv) antibodies carrying the kappa light-chain variable domain or expressing the V(H)III (variable domain of the heavy chain of Ig) determinant, were efficiently purified on immobilized protein LA. As judged by surface plasmon resonance (SPR), protein LA showed enhanced affinity for all tested ligands, including several scFv antibodies, compared with proteins L and A alone. SPR analysis also demonstrated that binding of a ligand to one of the components in protein LA did not affect the ability of the hybrid protein to interact simultaneously with a ligand for the other component. The antigen-binding capacity of a kappa-expressing scFv antibody was unaffected by the interaction with protein LA, whereas the binding of a V(H)III-expressing scFv antibody to its antigen was, unexpectedly, blocked by protein A and protein LA. Together, these data demonstrate that protein LA represents a highly versatile Ig-binding molecule.
Svensson
H G
HG
Department of Medical Microbiology, University of Lund, Sweden.
Hoogenboom
H R
HR
Sjöbring
U
U
D000906:Antibodies / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics*; D015374:Biosensing Techniques; D006801:Humans; D007128:Immunoglobulin Fragments / Q000378:metabolism*; D007145:Immunoglobulin kappa-Chains / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D013205:Staphylococcal Protein A / Q000235:genetics*; D020349:Surface Plasmon Resonance
D000906:Antibodies; D001426:Bacterial Proteins; C055047:Ig L-binding protein, Peptostreptococcus; D007128:Immunoglobulin Fragments; D007145:Immunoglobulin kappa-Chains; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A