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.
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.
Nuttall
S D
SD
CSIRO Health Sciences and Nutrition, Parkville, Victoria, Australia. Stewart.Nuttall@hsn.csiro.au
Hattarki
M
M
Guthrie
R E
RE
Hudson
P J
PJ
Kortt
A A
AA
D000123:Acetyltransferases / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000906:Antibodies / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D001483:Base Sequence; D015374:Biosensing Techniques; D003001:Cloning, Molecular; D019281:Dimerization; D004352:Drug Resistance, Microbial / Q000235:genetics; D000939:Epitopes / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009842:Oligopeptides; D010455:Peptides / Q000276:immunology / Q000378:metabolism; D011392:Proline
D000906:Antibodies; D001426:Bacterial Proteins; D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; C099353:phleomycin resistance protein, Streptoalloteichus hindustanus; C085706:FLAG peptide; D011392:Proline; C087954:bleomycin N-acetyltransferase; D000123:Acetyltransferases