Efficient heterologous expression and one-step purification of fully active c-terminal histidine-tagged uridine monophosphate kinase from Mycobacterium tuberculosis.
Tuberculosis has long been recognized as one of the most significant public health problems. Finding novel antituberculous drugs is always a necessary approach for controlling the disease. Mycobacterium tuberculosis pyrH gene (Rv2883c) encodes for uridine monophosphate kinase (UMK), which is a key enzyme in the uridine nucleotide interconversion pathway. The enzyme is essential for M. tuberculosis to sustain growth and hence is a potential drug target. In this study, we have developed a rapid protocol for production and purification of M. tuberculosis UMK by cloning pyrH (Rv2883c) of M. tuberculosis H37Rv with the addition of 6-histidine residues to the C-terminus of the protein, and expressing in E. coli BL21-CodonPlus (DE3)-RIPL using an auto-induction medium. The enzyme was efficiently purified by a single-step TALON cobalt affinity chromatography with about 8 fold increase in specific activity, which was determined by a coupled assay with the pyruvate kinase and lactate dehydrogenase. The molecular mass of monomeric UMK was 28.2 kDa and that of the native enzyme was 217 kDa. The enzyme uses UMP as a substrate but not CMP and TMP and activity was enhanced by GTP. Measurements of enzyme kinetics revealed the kcat value of 7.6 +/- 0.4 U mg(-1) or 0.127 +/- 0.006 sec(-1).The protocol reported here can be used for expression of M. tuberculosis UMK in large quantity for formulating a high throughput target-based assay for screening anti-tuberculosis UMK compounds.
Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand.
Praweenuch
P
Penpassakarn
Pimchai
P
Chaiyen
Prasit
P
Palittapongarnpim
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006639:Histidine / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006639:Histidine; C023276:uridine monophosphate kinase; D009703:Nucleoside-Phosphate Kinase