Cloning, expression and purification of Rv2623
The coding sequence of rv2623 was PCR-amplified from M. tuberculosis Erdman genomic DNA and subcloned into the expression vector pQE80L (Qiagen, Inc.), which encodes an N-terminal His6-tag, producing the plasmid pQE-rv2623 (Text S1). Expression was carried out following isopropyl beta-D-thiogalactoside (IPTG) induction of BL21 E. coli transformed with pQE-rv2623. His6-Rv2623 was then affinity-purified to homogeneity from BL21 cell lysates using Ni-NTA agarose (Qiagen, Inc.) according to the manufacturer's instructions. For crystallization, purified Rv2623 was concentrated to 12 mg/ml using a 10 kDa Molecular Weight Cut Off (MWCO) centrifugal filter (Amicon), and frozen at −80°C.