tuberculosis H37Rv strain (laboratory strain: ATCC 25618) were the sources of the leuA gene with 14 and 2 copies, respectively, of the 57 bp tandem repeat [25]. E. coli was grown in Luria-Bertani (LB) medium. M. tuberculosis was grown on Middlebrook 7H11 agar supplemented with 10% Middlebrook OADC [Oleic acid Albumin Dextrose Catalase] Enrichment (Difco BBL). DNA manipulations Standard protocols for DNA manipulation, DNA transformation,

DNA sequencing and PCR amplification were performed as previously described [29, 30]. M. tuberculosis genomic DNA was prepared as previously described [31]. Cloning of the leuA gene containing 14 copies of the repeat units by PCR amplification Primer PD98059 research buy design: two primers, leu44 (5′-GGA ATT CCA TAT GAC AAC TTC TGA ATC

GCC C-3′) and leu66 (5′ -CGC GGA TCC CTA GCG TGC CGC CCG GTT GAC-3′) [4], which flank the 5′ and 3′ ends of the leuA gene, were designed to include NdeI and BamHI Compound Library research buy recognition sites to facilitate the cloning of the leuA gene into pET15b (Novagen). We used 50 μl reaction mixtures containing 50 ng DNA template, 0.2 mM each dNTP, 1 mM each primer, 1.25 mM MgCl2, 2 units Taq DNA polymerase, 10 mM Tris-HCl (pH 8.3), 50 mM KCl and 0.1% Tween20 for PCR. Reactions were denatured at 94°C for 2 min and then cycled through 30 rounds of denaturation at 94°C for 30 sec, annealing at 62°C for 2 min, and extension at 72°C for 2 min. These cycles were followed with a final Adenosine triphosphate cycle at 72°C for 10 min. PCR products from strain 731 were purified using a PCR purification kit (QIAGEN, Valencia, CA, USA), digested with NdeI and BamHI, ligated to compatible sites in pET15b and transformed into E. coli DH5α. Correct clones were identified by colony-PCR and subsequently confirmed by restriction enzyme digestion and DNA sequencing. The PP1 and PP2 primers (PP1: 5′-tac tac gag cac gcg atg a-3′,

PP2: 5′-GTG ATT GAC GGT GCG AT-3′), which flanked the tandem repeats, were used to sequence the cloned genes. The recombinant plasmids were then transformed into E. coli BL21 (λDE3). Protein expression E. coli BL21 (λDE3) cells harboring the recombinant plasmids were grown at 37°C in LB medium supplemented with 100 μg/ml of ampicillin until the culture reached mid log phase (~0.3–0.4 OD600). IPTG was added to the culture to a final concentration of 0.5 mM. The culture was incubated at 20°C with shaking overnight. The bacterial cells were harvested by centrifugation, washed once with 50 mM Tris-HCl, pH 7.0, and stored at -70°C until use. Protein purification One milligram of cells (wet weight) from 200 ml of culture media was resuspended in 1 ml lysis buffer (10 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and lysed by sonication. The cell lysate was centrifuged at 10,000 g for 30 min to separate the soluble and insoluble fractions. Cleared lysate containing the His6-tagged protein was transferred to a tube containing 0.