Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain.

Small protein domains, capable of specific binding to different target proteins have been selected using combinatorial approaches. These binding proteins, called affibodies, were designed by randomization of 13 solvent-accessible surface residues of a stable alpha-helical bacterial receptor domain Z, derived from staphylococcal protein A. Repertoires of mutant Z domain genes were assembled and inserted into a phagemid vector adapted for monovalent phage display. Two libraries, each comprising approximately 4 x 10(7) transformants, were constructed using either an NN(G/T) or an alternative (C/A/G)NN degeneracy. Biopanning against the target proteins Taq DNA polymerase, human insulin, and a human apolipoprotein A-1 variant, showed that in all cases significant enrichments were obtained by the selection procedures. Selected clones were subsequently expressed in Escherichia coli and analyzed by SDS-PAGE, circular dichroism spectroscopy, and binding studies to their respective targets by biospecific interaction analysis. The affibodies have a secondary structure similar to the native Z domain and have micromolar dissociation constants (KD) for their respective targets.