Affilin-novel binding molecules based on human gamma-B-crystallin, an all beta-sheet protein

J Mol Biol. 2007 Sep 7;372(1):172-85. doi: 10.1016/j.jmb.2007.06.045. Epub 2007 Jun 22.

Abstract

The concept of novel binding proteins as an alternative to antibodies has undergone rapid development and is now ready for practical use in a wide range of applications. Alternative binding proteins, based on suitable scaffolds with desirable properties, are selected from combinatorial libraries in vitro. Here, we describe an approach using a beta-sheet of human gamma-B-crystallin to generate a universal binding site through randomization of eight solvent-exposed amino acid residues selected according to structural and sequence analyses. Specific variants, so-called Affilin, have been isolated from a phage display library against a variety of targets that differ considerably in size and structure. The isolated Affilin variants can be produced in Escherichia coli as soluble proteins and have a high level of thermodynamic stability. The crystal structures of the human wild-type gamma-B-crystallin and a selected Affilin variant have been determined to 1.7 A and 2.0 A resolution, respectively. Comparison of the two molecules indicates that the human gamma-B-crystallin tolerates amino acid exchanges with no major structural change. We conclude that the intrinsically stable and easily expressed gamma-B-crystallin provides a suitable framework for the generation of novel binding molecules.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carrier Proteins / chemical synthesis*
  • Carrier Proteins / chemistry
  • Carrier Proteins / isolation & purification*
  • Carrier Proteins / metabolism
  • Cattle
  • Estradiol / metabolism
  • Feasibility Studies
  • Humans
  • Immunoglobulin G / metabolism
  • Models, Molecular
  • Nerve Growth Factor / metabolism
  • Peptide Library
  • Protein Binding
  • Protein Engineering / methods*
  • Protein Precursors / metabolism
  • Protein Structure, Secondary
  • Substrate Specificity
  • Testosterone / metabolism
  • gamma-Crystallins / chemistry*

Substances

  • Carrier Proteins
  • Immunoglobulin G
  • Peptide Library
  • Protein Precursors
  • crystallin, gammaB
  • gamma-Crystallins
  • pro-nerve growth factor, human
  • Testosterone
  • Estradiol
  • Nerve Growth Factor

Associated data

  • PDB/2JDF
  • PDB/2JDG