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.
Nord
K
K
Department of Biochemistry and Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden.
Gunneriusson
E
E
Ringdahl
J
J
Ståhl
S
S
Uhlén
M
M
Nygren
P A
PA
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
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