Production and epitope characterization of mAbs specific for translation factor IF1.
Initiation of protein synthesis in bacteria relies on the presence of three translation initiation factors, of which translation initiation factor IF1 is the smallest having a molecular weight of only 8.2kDa. In addition to its function in this highly dynamic process, the essential IF1 protein also functions as an RNA chaperone. Despite extensive research, the exact function of IF1 in translation initiation has not yet been determined, and the research in the function of the factor has in some areas been impeded by the lack of monoclonal antibodies specific for this protein. Several attempts to induce immune response in mice with wild-type IF1 for the production of antibodies have failed. We have now succeeded in producing monoclonal antibodies specific for IF1 by applying a new immunization strategy involving an antigen combination of IF1 coupled to glutathione S-transferase (GST) and a recombinant dimer of IF1. This resulted in the generation of 6 IgG, 2 IgM, and 1 IgA anti-IF1 antibodies, which can be used in ELISA screening and Western immunoblots. We also provide a mapping of the functional epitopes of the generated anti-IF1 monoclonal antibodies by screening the antibodies for binding to IF1 proteins mutated at single amino acid positions.
Rasmussen
Louise Carøe Vohlander
LC
Department of Molecular Biology, Aarhus University, DK-8000 Aarhus C, Denmark.
Jensen
Janni Mosgaard
JM
Croitoru
Victor
V
Sperling-Petersen
Hans Uffe
HU
Mortensen
Kim Kusk
KK
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D004926:Escherichia coli / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D039861:Prokaryotic Initiation Factor-1 / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis
D000911:Antibodies, Monoclonal; D039861:Prokaryotic Initiation Factor-1; D011994:Recombinant Proteins