A phage display system for studying the sequence determinants of protein folding

Protein Sci. 1995 Jun;4(6):1108-17. doi: 10.1002/pro.5560040609.

Abstract

We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First, the phage selection procedure strongly selects against a point mutation in protein L that disrupts folding but is not in the IgG binding interface. Second, variants recovered from a library in which the first third of protein L was randomized are properly folded. The degree of sequence variation in the selected population is striking: the variants have as many as nine substitutions in the 14 residues that were mutagenized. The approach provides a selection for "foldedness" that is potentially applicable to any small binding protein.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism*
  • Bacteriophage M13 / genetics*
  • Base Sequence
  • Circular Dichroism
  • Gene Library
  • Genetic Vectors / genetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis*
  • Plasmids / genetics
  • Protein Denaturation
  • Protein Folding*
  • Recombinant Proteins / metabolism
  • Selection, Genetic
  • Structure-Activity Relationship

Substances

  • Bacterial Proteins
  • Ig L-binding protein, Peptostreptococcus
  • Recombinant Proteins