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 bin ding proteins, Galled affibodies, were designed by randomization of 13 solvent-accessible surface residues of a stable alpha-helical bacteri al receptor domain Z, derived from staphylococcal protein A. Repertoir es of mutant Z domain genes were assembled and inserted into a phagemi d vector adapted for monovalent phage display. Two libraries, each com prising approximately 4x10(7) transformants, were constructed using ei ther an NN(G/T) or an alternative (C/A/G)NN degeneracy Biopanning agai nst: the target proteins Tag DNA polymerase, human insulin, and a huma n apolipoprotein A-1 variant, showed that in all cases significant enr ichments were obtained by file selection procedures. Selected clones w ere subsequently expressed in Escherichia coli and analyzed by SDS-PAG E, circular dichroism spectroscopy, and binding studies to their respe ctive targets by biospecific interaction analysis. The affibodies have a secondary structure similar to the native Z domain and have micromo lar dissociation constants (K-D) for their respective targets.