The Fibronectin Type III Domain as a Scaffold for Novel Binding Proteins

J Mol Biol. 1998 Dec 11;284(4):1141-51. doi: 10.1006/jmbi.1998.2238.

Abstract

The fibronectin type III domain (FN3) is a small autonomous folding unit which occurs in many animal proteins involving in ligand binding. The beta-sandwich structure of FN3 closely resembles that of immunoglobulin domains. We have prepared a phage display library of FN3 in which residues in two surface loops were randomized. We have selected mutant FN3s which bind to a test ligand, ubiquitin, with significant affinities, while the wild-type FN3 shows no measurable affinity. A dominant clone was expressed as a soluble protein and its properties were investigated in detail. Heteronuclear NMR characterization revealed that the selected mutant protein retains the global fold of FN3. It also has a modest conformational stability despite mutations at 12 out of 94 residues. These results clearly show the potential of FN3 as a scaffold for engineering novel binding proteins.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Bacteriophage M13 / genetics
  • Base Sequence
  • Binding Sites / genetics
  • Carrier Proteins / chemistry*
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • DNA, Recombinant / genetics
  • Fibronectins / chemistry*
  • Fibronectins / genetics
  • Fibronectins / metabolism*
  • Humans
  • In Vitro Techniques
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis
  • Protein Conformation
  • Protein Engineering
  • Protein Folding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism

Substances

  • Carrier Proteins
  • DNA, Recombinant
  • Fibronectins
  • Recombinant Proteins