Identification of epitope recognized by an anti-c-myc monoclonal antibody that cross-reacts with E. coli sigma factor using phage display libraries.
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.


OBJECTIVE
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.


METHODS
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.


CONCLUSIONS
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.
Ikegaki
N
N
Division of Oncology, Children's Hospital of Philadelphia, Abramson Research Center, PA 19104-4318, USA. ikegaki@kermit.oncol.chop.edu
Tang
X X
XX
Kay
B K
BK
Kennett
R H
RH
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D003090:Coliphages / Q000235:genetics; D003429:Cross Reactions / Q000276:immunology*; D017931:DNA Primers / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000276:immunology*; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology; D015723:Gene Library; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010455:Peptides / Q000235:genetics / Q000276:immunology; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000276:immunology*; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017421:Sequence Analysis; D012808:Sigma Factor / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D010455:Peptides; D016271:Proto-Oncogene Proteins c-myc; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases