Utilization of the Streptoalloteichus hindustanus resistance determinant ShBle as a protein framework: effect of mutation upon ShBle dimerization and interaction of C-terminal displayed peptide epitopes

J Protein Chem. 1999 Nov;18(8):813-21. doi: 10.1023/a:1020618910455.

Abstract

We have selected the Streptoalloteichus hindustanus bleomycin-resistance protein ShBle, a 28-kDa homodimer, as a scaffold for the display of bioactive peptides and other peptide epitopes. To create a monomeric scaffold, we investigated the effect of mutating residue proline 9 to glycine. This residue plays a critical role in ShBle dimerization by affecting the position of the eight N-terminal residues which secure the interaction between the monomeric subunits. We demonstrate that this mutation weakens the dimerization interaction, resulting in establishment of a stable equilibrium between monomeric and dimeric ShBle species in solution. Circular dichroism and SDS-PAGE data indicate that the Pro9Gly mutation does not disrupt the structure of the molecule. Production of a fully monomeric form of ShBle required complete removal of the eight-residue N-terminal peptide, and the interaction across the now solvent-exposed hydrophobic interface of the ShBle monomer was insufficient to drive dimerization. To demonstrate efficient display of epitope tags on the ShBle protein, we displayed dual-octapeptide FLAG tags at the protein C-terminus. These additions did not interfere with protein folding or activity. The resulting ShBle scaffold was used to compare the efficiency of two commercial FLAG-specific antibodies by biosensor.

Publication types

  • Comparative Study

MeSH terms

  • Acetyltransferases / chemistry
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Antibodies / metabolism
  • Bacterial Proteins / genetics
  • Bacterial Proteins / immunology*
  • Bacterial Proteins / metabolism*
  • Base Sequence
  • Biosensing Techniques
  • Cloning, Molecular
  • Dimerization
  • Drug Resistance, Microbial / genetics
  • Epitopes / metabolism*
  • Escherichia coli / genetics
  • Molecular Sequence Data
  • Mutation
  • Oligopeptides
  • Peptides / immunology
  • Peptides / metabolism
  • Proline

Substances

  • Antibodies
  • Bacterial Proteins
  • Epitopes
  • Oligopeptides
  • Peptides
  • phleomycin resistance protein, Streptoalloteichus hindustanus
  • FLAG peptide
  • Proline
  • bleomycin N-acetyltransferase
  • Acetyltransferases