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Updated: Jul 5, 2026
Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments
Published on: October 15, 2016
Identification of epitope recognized by an anti-c-myc monoclonal antibody that cross-reacts with E. coli sigma factor
1Division of Oncology, Children's Hospital of Philadelphia, Abramson Research Center, PA 19104-4318, USA. ikegaki@kermit.oncol.chop.edu
Background:
During the epitope mapping of monoclonal antibodies specific for myc proteins, two E. coli proteins cross-reactive with an anti-c-myc monoclonal antibody (MYC-X-5/1) were identified. One of the proteins is approximately 90 kDa and the other is over 150 kDa in apparent molecular mass. The molecular masses of these cross-reactive proteins suggested that they may be subunits of E. coli RNA polymerase.
Objectives:
We have investigated whether or not the proteins cross-reactive with MYC-X-5/1 are subunits of E. coli RNA polymerase. In addition, we have attempted to determine the epitope of MYC-X-5/1.
Study Design:
The reactivity of MYC-X-5/1 antibody was tested against highly purified E. coli RNA polymerase holo-enzyme preparations and the cell lysate made from E. coli carrying a multi-copy plasmid with an insert of the rpoD gene, the structural gene for the E. coli sigma subunit. The epitope of MYC-X-5/1 was determined by use of phage display of random peptide libraries.
Results:
On immunoblotting assays, MYC-X-5/1 reacted with the 90-kDa protein in the E. coli RNA polymerase preparations and with the 90-kDa protein over-expressed in E. coli carrying the plasmid with the rpoD insert. In addition, we have deduced the epitope of the MYC-X-5/1 antibody to be residues 235-245 of the human c-myc protein. A highly similar sequence to this was also identified in residues 62-72 of the sigma subunit of E. coli RNA polymerase.
Conclusion:
These data demonstrated that the 90-kDa protein cross-reactive with MYC-X-5/1 is the sigma subunit of E. coli RNA polymerase. Furthermore, this study shows that random peptide libraries displayed on filamentous phage are useful tools for epitope mapping and defining cross-reactivities of monoclonal antibodies.
