br Materials and methods Bacterial strains

Materials and methods Bacterial strains and growth conditions. Escherichia coli TOP10 and E. coli BL21Star (DE3) (ThermoFisher, Waltham, MA) were grown at 37 °C in Luria broth (LB) or LB-agar containing kanamycin (50 μg/ml). Bioinformatic analyses. The amino transporters sequence of Rv2477c was obtained from Tuberculist [11]. Other amino acid sequences were obtained from UniProt (http://www.uniprot.org/). ClustalOmega (http://www.ebi.ac.uk/Tools/msa/clustalo/) was used to align the sequences. BoxShade (http://www.ch.embnet.org/software/BOX_form.html) was used to shade similar and identical amino acids in the aligned sequences. The information on the genetic organization of the locus around Rv2477c and its orthologs in mycobacteria was obtained from Genolist (http://genolist.pasteur.fr) and KEGG (http://www.kegg.jp). Cloning of Rv2477c and Rv2477c-EQmutant proteins. Rv2477c was amplified from the genomic DNA of Mycobacterium tuberculosis H37Rv by polymerase chain reaction using the following primers (Forward: 5′-CACCATGGCTGAGTTCATCTACACGATG-3’; Reverse: 5′-TTAGCCGCGCGTCAGCTTGCG-3′). The amplified product was cloned into pET200 D-TOPO plasmid (ThermoFisher, Waltham, MA) and sequence integrity was verified by DNA sequencing. The Rv2477c-EQ2 mutant protein containing glutamate to glutamine substitutions at amino acid positions 185 and 468 in the two Walker B motifs on the Rv2477c protein was generated using the Rv2477c-pET200 plasmid construct as template by site-directed mutagenesis using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA) following the manufacturer's protocols in two stages. The primers used in the first stage to generate the E to Q mutation at residue 185 were: SDM553F: 5′-GTTGCTCGACCAGCCGACCAA-3’; SDM553R: 5′-AACAGGTCGGGTTTGGAC-3’. The mutated plasmid construct was then used as template to generate the second mutation (corresponding to amino acid residue 468 on Rv2477c) using the following primers: SDM1402F: 5′-TCTGCTCGACCAACCGACGAAC-3’; SDM1402R: 5′-ATCAGGTTGCCGCCCTGT-3’. The mutations were confirmed by DNA sequencing of the plasmid construct. Expression and purification of Rv2477c and Rv2477c-EQproteins. The Rv2477c-pET200 or Rv2477c-EQ2-pET200 plasmid construct was used to transform chemically competent E. coli BL21 Star (DE3). A 300 ml LB culture containing 0.4 M sucrose was grown at 37 °C until log-phase (OD600 ∼0.6). The culture was heat-shocked at 42 °C for 20 min (to enhance soluble protein production) and after cooling to 22 °C, expression of Rv2477c protein was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG; IBI Scientific, Peosta, IA) for 16 h at 22 °C with shaking. Protein expression was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. Cells from the induced culture were lysed by ultrasonication on ice in the presence of 0.2 mM phenylmethylsulfonyl fluoride. The Rv2477c protein was purified from the 16,000 × g supernatant of the lysate by cobalt-affinity chromatography using the TALON cobalt-affinity resin (Takara Bio USA, Inc., Mountain View, CA). Following binding of the protein at 22 °C to 1 ml bed-volume of TALON resin equilibriated with equilibriation/wash buffer (50 mM Tris-HCl pH 7.0, 500 mM NaCl), the column flow-through was collected. The resin was washed with 10 bed-volumes of equilibriation/wash buffer containing 5 mM imidazole followed by 10 bed-volumes of equilibriation/wash buffer containing 10 mM imidazole. Elution of bound Rv2477c protein from the resin was done using 5 bed-volumes equilibriation/wash buffer containing 50 mM imidazole. The fractions were checked for purity by 12% SDS-PAGE followed by Coomassie staining and fractions containing Rv2477c were pooled, concentrated and buffer-exchanged into 50 mM Tris-HCl pH 7.0, 300 mM NaCl, 10% glycerol by six cycles of ultra-filtration using a 50,000 molecular weight cut-off (MWCO) centrifugal membrane filter (EMD Millipore, Billerica, MA). The purified protein was quantitated by Bradford protein assay (Bio-Rad, Hercules, CA) and purity of the protein was confirmed by SDS-PAGE and Coomassie staining.