title: Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain.
abstract: 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.
authors.affiliation: Department of Biochemistry and Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden.
authors.forename: K
authors.initials: K
authors.lastname: Nord
authors.forename: E
authors.initials: E
authors.lastname: Gunneriusson
authors.forename: J
authors.initials: J
authors.lastname: Ringdahl
authors.forename: S
authors.initials: S
authors.lastname: Ståhl
authors.forename: M
authors.initials: M
authors.lastname: Uhlén
authors.forename: P A
authors.initials: PA
authors.lastname: Nygren
mesh_terms: D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D015374:Biosensing Techniques; D002942:Circular Dichroism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D015195:Drug Design; D004926:Escherichia coli; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011971:Receptors, Immunologic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019914:Taq Polymerase
chemical_list: D016632:Apolipoprotein A-I; D007141:Immunoglobulin Fc Fragments; D007328:Insulin; D019151:Peptide Library; D011971:Receptors, Immunologic; C028706:Staph protein A receptor; D013205:Staphylococcal Protein A; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase