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

We have selected the Streptoalloteichus hindustanus bleomycin-resistance pr otein ShBle, a 28-kDa homodimer, as a scaffold for the display of bioactive peptides and other peptide epitopes. To create a monomeric scaffold, we in vestigated the effect of mutating residue proline 9 to glycine. This residu e plays a critical role in ShBle dimerization by affecting the position of the eight N-terminal residues which secure the interaction between the mono meric subunits. We demonstrate that this mutation weakens the dimerization interaction, resulting in establishment of a stable equilibrium between mon omeric and dimeric ShBle species in solution. Circular dichroism and SDS-PA GE data indicate that the Pro9Gly mutation does not disrupt the structure o f the molecule. Production of a fully monomeric form of ShBle required comp lete removal of the eight-residue N-terminal peptide, and the interaction a cross the now solvent-exposed hydrophobic interface of the ShBle monomer wa s insufficient to drive dimerization. To demonstrate efficient display of e pitope tags on the ShBle protein, we displayed dual-octapeptide FLAG tags a t the protein C-terminus. These additions did not interfere with protein fo lding or activity. The resulting ShBle scaffold was used to compare the eff iciency of two commercial FLAG-specific antibodies by biosensor.