Recognition sequences of the restriction enzymes NdeI (CATATG) and XhoI (CTCGAG) are in italics. The PCR products were digested with NdeI and XhoI, ligated to appropriately digested expression plasmid pET23b with C-terminal histidine tag, INCB28060 cell line and transformed into E. coli DH5α. Transformants were selected with ampicillin. Plasmids were purified from the transformants and sequenced to confirm the presence of correct genes tagged at the 3′ end with 6 histidine codons, designated as pET23b-35c and pET23b-89 respectively. E. coli BL21(DE3) strain was transformed with pET23b-35c
and pET23b-89 to obtain strains BL21(DE3)-35c and BL21(DE3)-89 used for protein expression and from which the recombinant C-terminal histidine tagged proteins C-His-Rv2135c and C-His-Rv0489 were purified respectively. Expression and purification A liter of LB medium with 100 μg/ml ampicillin was inoculated with an overnight culture of BL21(DE3)-35c to a final OD600nm of about 0.03. The culture was incubated at 37°C with shaking speed
of 200 rpm until OD600nm reached about 0.6. The expression of the protein was then induced by the addition of IPTG to a final concentration of 0.4 mM. The culture was further incubated at 25°C at the shaking speed of 200 rpm for 8 hours. Cells were harvested by centrifugation at 3500 rpm at 4°C, washed with PBS pH 7.4 and stored at −20°C. Similar treatment of BL21(DE3)-89 Semaxanib was done and resulted in precipitation of expressed protein after lysis. In order to obtain C-His-Rv0489 in the soluble fraction of cell lysate, BL21(DE3)-89 was cultured in the www.selleck.co.jp/products/cobimetinib-gdc-0973-rg7420.html same media as above with the addition of 10% sucrose to OD600nm of about 0.03. After the OD600nm reached about 0.6, the expression of C-His-Rv0489
was induced with 0.03 mM of IPTG at 18°C overnight. Cells were harvested by centrifugation at 3500 rpm at 4°C, washed with PBS pH 7.4 and stored at −20°C. Frozen cells were thawed on ice and suspended in the lysis buffer (20 mM Tris–HCl pH 7.0, 100 mM NaCl, 1 mM PMSF, 5 mM imidazole). The suspended cells were lysed by sonication using Misonix Sonicator 3000 (Qsonica LLC, USA) with 30 sec pause intervals until a clear lysate was obtained. The lysate was centrifuged at 11,000 rpm at 4°C for 20 min. The supernates, which contained the expressed histidine tagged protein, C-His-Rv2135c and C-His-Rv0489, were separated from other soluble proteins by immobilized metal affinity chromatography (IMAC). Briefly, the crude extracts were applied to cobalt charged resin column (Talon® Superflow column, GE Healthcare, Sweden) pre-equilibrated with the wash buffer (20 mM Tris–HCl pH 7.0, 100 mM NaCl, 5 mM imidazole). The column was then washed with 4 volumes of the wash buffer. For C-His-Rv2135c, the progress of purification was Screening high throughput screening monitored by fast protein liquid chromatography (FPLC) using AKTA system (GE Healthcare, Sweden).