WT rv2466c was introduced into pET28B (Novagen),
and the resulting plasmid was transformed into BL21(DE3) competent
cells (Life technologies) with kanamycin selection (50 μg/mL).
Bacteria were grown initially at 37 °C while shaking, and protein
expression was induced using 0.75 mM IPTG at 16 °C for 18 h.
Cells were pelleted and lysed using a sonicator and His tagged protein
was purified with nickel chromatography and dialyzed into a 50 mM
Tris, pH 7.5, 50 mM NaCl solution. On some occasions, protein was
further purified by FPLC. Protein concentration was measured using
a BCA kit (Thermofisher). Purity and size of the protein was confirmed
using SDS-PAGE gel and Coomassie staining (Biorad) (Figure S8 ). Mutant proteins were constructed by introduction
of point mutations into the pET28B plasmid that contains the WT rv2466c using the Quick Change II site directed mutagenesis
kit (Agilent). For expression in mycobacteria, rv2466c was cloned into pMV261 (for episomal expression) or pMV306 (genomic
expression) plasmids under the control of the hsp60 promoter for constitutive
expression and selected by kanamycin.
and the resulting plasmid was transformed into BL21(DE3) competent
cells (Life technologies) with kanamycin selection (50 μg/mL).
Bacteria were grown initially at 37 °C while shaking, and protein
expression was induced using 0.75 mM IPTG at 16 °C for 18 h.
Cells were pelleted and lysed using a sonicator and His tagged protein
was purified with nickel chromatography and dialyzed into a 50 mM
Tris, pH 7.5, 50 mM NaCl solution. On some occasions, protein was
further purified by FPLC. Protein concentration was measured using
a BCA kit (Thermofisher). Purity and size of the protein was confirmed
using SDS-PAGE gel and Coomassie staining (Biorad) (
of point mutations into the pET28B plasmid that contains the WT rv2466c using the Quick Change II site directed mutagenesis
kit (Agilent). For expression in mycobacteria, rv2466c was cloned into pMV261 (for episomal expression) or pMV306 (genomic
expression) plasmids under the control of the hsp60 promoter for constitutive
expression and selected by kanamycin.