title: Identification of protein partners in mycobacteria using a single-step affinity purification method.
abstract: Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spread and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus vital. We have implemented improved screening methods for protein-protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method can be efficiently applied to both medium- and high-throughput studies aiming to characterize protein-protein interaction networks of tubercle bacilli. Of the 4 tested epitopes FLAG, enhanced green fluorescent protein (eGFP), protein A and haemagglutinin, the eGFP tag was found to be most useful on account of its easily monitored expression and its ability to function as a simultaneous tool for subcellular localization studies. It presents a relatively low background with cost-effective purification. RNA polymerase subunit A (RpoA) was used as a model for investigation of a large protein complex. When used as bait, it co-purified with all remaining RNA polymerase core subunits as well as many accessory proteins. The amount of RpoA strongly correlated with the amount of quantification peptide used as part of the tagging system in this study (SH), making it applicable for semi-quantification studies. Interactions between the components of the RpoA-eGFP protein complex were further confirmed using protein cross-linking. Dynamic changes in the composition of protein complexes under induction of UV damage were observed when UvrA-eGFP expressing cells treated with UV light were used to co-purify UvrA interaction partners.
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
authors.forename: Przemysław
authors.initials: P
authors.lastname: Płociński
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Laubitz
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
authors.forename: Dominik
authors.initials: D
authors.lastname: Cysewski
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
authors.forename: Krystian
authors.initials: K
authors.lastname: Stoduś
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Kowalska
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland; Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Dziembowski
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D009161:Mycobacterium / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins