Characterization of an anti-FLAG antibody binding protein in V. cholerae
Introduction
Affinity tags have been developed to improve production of recombinant proteins and well-characterized epitope-tag systems have become major tools for life science research. Antibodies for the detection of such affinity tags are commercially available without the need for costly and time-intensive generation of custom antibodies. Relatively small epitope tags such as His, c-Myc, Strep, HA (hemagglutinin), Spot, T7, Glu-Glu and particularly the FLAG-tag are widely used for the detection of fusion proteins to facilitate protein abundance measurements, purification and interaction studies, in vitro and in cell culture [[1], [2], [3], [4], [5], [6], [7]]. Their short, linear recognition motifs rarely affect the properties of the heterologous protein of interest and are usually very specific for their respective primary antibodies. The FLAG system has been used in a variety of cell types, including bacteria [8,9], yeast [10,11], and mammalian cells [12,13]. The FLAG-tag system utilizes a short, hydrophilic eight amino acid peptide that is fused to the protein of interest at the C- or N- terminus of the target protein [14].
Protein quantification via Western Blot and identification of binding complexes by immunoprecipitation rely on the specificity of the antibody for binding its cognate epitope tag and, for the most common tags, non-specific background binding is generally low. Where non-specific background does occur, it is often ignored or used as an internal loading control, assuming that background detection is stable over many conditions tested (e.g., when comparing protein abundances during a growth time course).
Sometimes, however, non-specific signals confound analysis of the intended target signals. For example, the Fur (Fe2+ uptake transcriptional regulator) subfamily transcription factors (Fur-Fe2+; PerR-Fe2+; Mur-Mn2+; Nur-Ni2+; Zur-Zn2+ and Irr-heme), which are well-conserved in bacteria, contain at least 3 or 7 direct repeated histidines and thus contaminate 6xHis tag fusion protein purification after binding to Ni-NTA resin [15].
Here, we report a FLAG-reactive protein in V. cholerae, the causative agent of cholera disease. We identified this protein as an outer membrane chitoporin that contains an amino acid sequence with striking similarity to the 3xFLAG tag. Our data should be informative for researchers using the FLAG tag in Vibrio cholerae.
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Bacterial strains and culture conditions
All V. cholerae strains used in this study (see Table S1) are derivatives of wild type El Tor strain N16961. Vibrio cholerae cells were grown in Lysogeny Broth (LB) medium supplemented with 200 μg/ml streptomycin sulfate (TCI, cat# S0585) at 37 °C with shaking at 200 rpm. E. coli DH5α λpir was used for routine DNA cloning. Sub-cloning was conducted in E. coli SM10 λpir for conjugal gene transfer into V. cholerae. E. coli BL21 DE3/pLysS (Novagen) and its derivative strain, Rosetta-gami2
Vibrio cholerae encodes a FLAG-antibody-reactive protein, which is differentially regulated by growth phase
During Western Blotting experiments with anti-Flag antibody aimed at mapping the growth-phase-dependent expression pattern of a 3xFLAG-tagged protein (Fig. 1A) in V. cholerae, we noticed a background band at around ∼36 kDa (Fig. 1B). We at first hypothesized that this band represented a product of proteolytic degradation of our target protein. However, the same band appeared as background in several other strains carrying 2 different FLAG-tagged proteins, as well as in an untagged wild-type
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
Research in the Dörr laboratory is supported by National Institutes of Health (NIH) grants R01AI143704 and R01GM130971. Research in the Smolka lab is funded by NIH grants R01HD095296 and R01GM123018.
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