Production and epitope characterization of mAbs specific for translation factor IF1 Louise Carøe Vohlander Rasmussen a , Janni Mosgaard Jensen a , Victor Croitoru b , Hans Uffe Sperling-Petersen a , Kim Kusk Mortensen a, * a Department of Molecular Biology, Aarhus University, DK-8000 Aarhus C, Denmark b Department of Genetics, Microbiology and Toxicology, Stockholm University, S-10691 Stockholm, Sweden Received 24 September 2007 Available online 4 October 2007 Abstract 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 8.2 kDa. In addition to its function in this highly dynamic process, the essen- tial 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 anti- bodies 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. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Monoclonal antibody; Translation initiation factor IF1; Immunization; Glutathione S-transferase; Epitope mapping; IF1 mutants Bacterial translation initiation factor IF1 is a small 8.2 kDa protein, which is involved in initiation of protein synthesis [1]. It has been shown to influence several steps in this process. IF1 stimulates formation of the 30S pre-ini- tiation complex [2–4] and inhibits premature docking of 50S subunits to 30S subunits lacking initiator tRNA [5]. Being positioned at the ribosomal A-site, IF1 occludes tRNAs from this site, accelerates binding of initiator tRNA to the P-site [6] and enhances the accuracy of initiator tRNA selection [7]. Removal of IF1 reduces initiation effi- ciency seven-fold as shown by kinetic studies [5], so IF1 is clearly an important factor even though a single specific function has not yet been assigned to it. IF1 also displays RNA chaperone activity [6,8]. During cold shock conditions the level of IF1 is moderately increased [9], and IF1 mutants confer cold sensitivity to cells [10]. Furthermore, the RNA chaperone activity has been linked to transcription antitermination [8], an activity also ascribed to cold shock proteins [11]. Mutating the amino acid residue responsible for the IF1 transcription antitermination activity does not affect cell viability, why it has been presumed that the essential function of IF1 is in translation and not in cold shock response. However, recent results indicate an essential role for IF1 during cold adaptation [12]. The structure of bacterial IF1 was determined by NMR spectroscopy in 1997 [13], and it revealed a remarkably rigid antiparallel five-stranded b-barrel. This structure is referred to as an oligonucleotide- or oligosaccharide-bind- ing fold (OB-fold) and is also found in cold shock proteins 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.09.120 * Corresponding author. Fax: +45 86 12 31 78. E-mail address: kkm@mb.au.dk (K.K. Mortensen). www.elsevier.com/locate/ybbrc Available online at www.sciencedirect.com Biochemical and Biophysical Research Communications 364 (2007) 72–78