Anti-idiotypic protein domains selected from protein A-based affibody libraries.
Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fc binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K(D)) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.
Department of Biotechnology, Royal Institute of Technology (KTH/SCFAB), Stockholm, Sweden.
Malin
M
Eklund
Lars
L
Axelsson
Mathias
M
Uhlén
Per-Ake
PA
Nygren
D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D001665:Binding Sites; D001709:Biotechnology; D007070:Immunoglobulin A / Q000378:metabolism; D008958:Models, Molecular; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D019914:Taq Polymerase / Q000378:metabolism
D000888:Antibodies, Anti-Idiotypic; D007070:Immunoglobulin A; D019151:Peptide Library; D013205:Staphylococcal Protein A; D019914:Taq Polymerase