Monoclonal antibodies against the Escherichia coli DNA repair protein RadA/Sms.
The RadA/Sms protein facilitates DNA repair in Escherichia coli cells damaged by UV radiation, X-rays, and chemical agents. However, the precise mechanism by which RadA/Sms aids DNA repair is unknown. Here we report the production of monoclonal antibodies (MAbs) specific for RadA/Sms for use in biochemical and physiological investigations. Histidine-tagged RadA/Sms (RadA-6xHis) was overproduced in E. coli BL21 cells transformed with the radA/sms coding region in plasmid pRSET A and purified by nickel affinity chromatography. Splenocytes from female BALB/c mice hyperimmunized with the purified protein were fused to SP2/0-Ag14 myeloma cells, and the resultant hybridomas were selected in HAT medium. MAbs were detected in hybridoma culture supernatants by indirect ELISA and Western blot analysis against purified RadA-6xHis. MAbs from four cell lines were further evaluated by Western blotting against peptide maps generated by endoproteinase Glu-C digestion of RadA-6xHis. Each of the four MAbs recognized a unique epitope on the fusion protein. Two of the MAbs (6F5 and 2A2) also detected wild-type (tagless) RadA/Sms produced from the pJS003 plasmid in E. coli K-12 cells. We anticipate that these antibodies will prove useful for the detection, isolation, and functional analysis of RadA/Sms.
Department of Microbiology/Immunology, A.T. Still University, Kirksville College of Osteopathic Medicine, 800 W. Jefferson Street, Kirksville, MO 63501, USA.
Nastassia C
NC
Richardson
Neil J
NJ
Sargentini
Vineet K
VK
Singh
Melissa K
MK
Stuart
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000276:immunology*; D005260:Female; D005822:Genetic Vectors; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D010449:Peptide Mapping; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D012697:Serine Endopeptidases / Q000378:metabolism; D014169:Transformation, Bacterial
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C491059:RadA protein, E coli; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase