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

Nat Biotechnol. 1997 Aug;15(8):772-7. doi: 10.1038/nbt0897-772.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Apolipoprotein A-I / metabolism
  • Binding Sites / genetics
  • Biosensing Techniques
  • Circular Dichroism
  • DNA-Directed DNA Polymerase / metabolism
  • Drug Design
  • Escherichia coli
  • Humans
  • Immunoglobulin Fc Fragments / metabolism*
  • Insulin / metabolism
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Library*
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Secondary
  • Receptors, Immunologic / chemistry
  • Receptors, Immunologic / genetics
  • Receptors, Immunologic / metabolism*
  • Sequence Alignment
  • Staphylococcal Protein A / chemistry
  • Staphylococcal Protein A / genetics
  • Staphylococcal Protein A / metabolism*
  • Taq Polymerase


  • Apolipoprotein A-I
  • Immunoglobulin Fc Fragments
  • Insulin
  • Peptide Library
  • Receptors, Immunologic
  • Staph protein A receptor
  • Staphylococcal Protein A
  • Taq Polymerase
  • DNA-Directed DNA Polymerase