Efficient Heterologous Expression and One-Step Purification of Fully Active C-Terminal Histidine-Tagged Uridine Monophosphate Kinase from Mycobacterium Tuberculosis
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SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH EFFICIENT HETEROLOGOUS EXPRESSION AND ONE-STEP PURIFICATION OF FULLY ACTIVE C-TERMINAL HISTIDINE-TAGGED URIDINE MONOPHOSPHATE KINASE FROM MYCOBACTERIUM TUBERCULOSIS Praweenuch Penpassakarn1, Pimchai Chaiyen2 and Prasit Palittapongarnpim3,4 1Department of Biotechnology, 2Department of Biochemistry and Center of Excellence in Protein Structure and Function, 3Department of Microbiology, Faculty of Science, Mahidol University, Bangkok; 4National Science and Technology Development Agency, Pathum Thani, Thailand Abstract. Tuberculosis has long been recognized as one of the most significant public health problems. Finding novel antituberculous drugs is always a neces- sary approach for controlling the disease. Mycobacterium tuberculosis pyrH gene (Rv2883c) encodes for uridine monophosphate kinase (UMK), which is a key en- zyme 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 ofM. 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 chroma- tography 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. Keywords: Mycobacterium tuberculosis, uridine monophosphate kinase, antituber- culous drugs, pyrimidine, protein purification INTRODUCTION become more worrisome than in the past mainly due to the emergence of multi- The widespread of tuberculosis, drug resistant strains (Dheda et al, 2010). caused by Mycobacterium tuberculosis, has The cases are often untreatable, signifying an urgent need for new antituberculous Correspondence: Prasit Palittapongarnpim, agents. Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok Current antituberculous drugs are 10400, Thailand. known to act on a limited number of drug Tel: 66 (0) 81 867 4202 targets, while recent investigations have E-mail: [email protected] shown hundreds of potential drug targets 1452 Vol 42 No. 6 November 2011 PURIFICATION OF M. TUBERCULOSIS URIDYLATE KINASE (Duncan, 2004). It is therefore necessary Pyrimidine monophosphate kinases to exploit these new drug targets for the found in bacteria are different from those development of new antituberculous of eukaryotes (Jones, 1980). There are compounds. three distinct prokaryotic pyrimidine Nucleotides are essential substrates monophosphate kinases, UMK, CMK and for bacterial growth, making interruption TMK, each specific for an individual py- of their metabolism being a viable strat- rimidylate. In contrast, phosphorylation egy in developing antituberculous drugs. of UMP and CMP in eukaryotic organisms Most bacteria obtain their nucleotides by is carried out by a bifunctional UMP/CMP both de novo biosynthesis and salvage kinase, which phosphorylates both UMP pathways. The pyrimidine de novo biosyn- and CMP with comparable efficiencies. thesis pathway appears to be nearly iden- The amino acid sequences of bacterial tical in all bacteria (Evrin et al, 2007). The CMK and eukaryotic UMP/CMP kinases six-membered pyrimidine is synthesized are similar to those of adenylate kinases, first from the reaction between carbamoyl while bacterial UMK is not homologous phosphate and aspartate, catalyzed by to any eukaryotic enzymes (Gagyi et al, aspartate transcarbamoylase. Dihydro- 2003). This suggests that UMK can serve orotase then catalyzes the closure of the as a potential anti-tuberculous drug target pyrimidine ring, which is subsequently as a chemical compound inhibiting M. oxidized to orotate by dihydroorotate tuberculosis enzyme should not affect the dehydrogenase using NAD+ as an electron human enzymes. Biochemical and cata- acceptor. Phosphoribosyl pyrophosphate lytic properties of the enzyme has recently (PRPP) is attached by the enzyme orotate been reported and the data are useful for phosphorybosyl transferase, resulting in developing specific inhibitors against the orotidylate, which is decarboxylated to enzyme (Rostirolla et al, 2011), but the yield uridine 5’-monophosphate (UMP), production yield is low (20 mg/2g of wet which is subsequently phosphorylated by cell paste and 21% enzyme yield). In or- uridine monophosphate kinase (UMK) der to improve the yield, which would be to yield uridine 5’-diphosphate (UDP). beneficial for high-throughput inhibitor UDP is converted to uridine 5’-triphos- screen assays, we report the development phate (UTP) by a non-specific nucleoside of an expression and purification protocol diphosphate kinase, and subsequently for production of fully active C-terminal forms cytidine 5’-triphosphate (CTP) by histidine-tagged M. tuberculosis UMP cytidylate synthetase. In the salvage path- (MtUMKHis6) in high yield. The purified way, pyrimidine bases are converted to enzyme had the expected biochemical uracil and subsequently to UMP by UMK. properties of M. tuberculosis UMK, indi- To our knowledge, no viable bacterial cating that the heterologously expressed deletion mutant of UMK has ever been enzyme can be used in high throughput reported. Mushegian and Koonin (1996) drug screening assays. identify pyrH as a member of the minimal gene set a highly conserved set of genes MATERIALS AND METHODS supposed to provide minimal essential functions of bacterial life. Its essentiality Strains and plasmids for M. tuberculosis has been experimen- M. tuberculosis H37Rv was used for tally confirmed (Robertson et al, 2007). genomic DNA extraction. E. coli XL-1 Vol 42 No. 6 November 2011 1453 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH blue (Invitrogen, Carlsbad, CA) was used compared. E. coli BL21-CodonPlus®(DE3)- for production of recombinant plasmid RIPL cells harboring pMtUMK were and E. coli BL21-CodonPlus®(DE3)-RIPL grown at 37ºC in 2xYT broth (16 g/l (Invitrogen, Carlsbad, Germany) for over- bacto-tryptone, 10 g/l yeast extract, and expression of pyrH. 0.5 g/l NaCl) supplemented with 50 µg/ml µ Cloning of M. tuberculosis pyrH coding kanamycin and 34 g/ml chlorampheni- sequence col. ZYP-5052 medium is composed of 10 g/l soluble enzymatic digest of casein, A 786 bp fragment containing the 5 g/l yeast extract, 50 mM Na HPO , 50 entire coding region of pyrH of M. tuber- 2 4 mM KH PO , 25 mM(NH ) SO , 2 mM culosis H37Rv was amplified by PCR using 2 4 4 2 4 MgSO , 0.5 g/l glucose and 2 g/l lactose pfu DNA polymerase with two primers, 4 and 0.2x trace metals (Studier, 2005) and containing NdeI and XhoI restriction sites supplemented with 50 µg/ml kanamy- (underlined), (pyrHF: 5’- CGTTAAC- cin and 34 µg/ml chloramphenicol. For CATATGACAGAGCCCGAT -3’, pyrHR: isopropyl-b-d-1-thiogalactopyranoside 5’-TTTCTCGAGGGTGGTGACCAGCGT (IPTG) induction, cells were grown until -3’). PCR was carried out in a 50 µl final the absorbance at 600 nm (OD ) reached volume containing 5 µl of 10x buffer 600 about 1.0. Then, induction was performed (Fermentas AB, Vilnius, Lituania), 25 µM by adding IPTG (Fermentas) to a final MgSO , 100 µM each dNTP, 0.5 µM each 4 concentration of 1 mM and was shaken primer, 2.0 U pfu DNA polymerase (Fer- at 20ºC, 250 rpm for 24 hours. For over- mentas) and 1 µg of M. tuberculosis H37Rv expression in auto-induction medium, DNA, using the following thermocycling E. coli BL21-CodonPlus®(DE3)-RIPL parameters: 5 minutes at 95ºC, 30 cycles of cells harboring pMtUMK were grown 95ºC for 60 seconds, 69.6ºC for 60 seconds at 37ºC in ZYP-5052. Cells were grown and 72ºC for 90 seconds with the final step until OD reached about 1.0 and then, of 5 minutes at 72ºC. PCR amplicons were 600 without adding IPTG, the culture was analyzed by electrophoresis in 1.5% (w/v) shaken at 20ºC, 250 rpm for 24 hours. agarose gel and stained with ethidium Bacteria were harvested by centrifugation bromide. The amplicon was inserted and at 4,000g for 10 minutes at 4ºC and stored ligated into pET-24b plasmid (EMB biosci- at -80ºC until used. Cell pastes from both ence, Madison, WI) at NdeI and XhoI sites methods of expression were suspended in using T4 DNA polymerase (Fermentas), 50 mM sodium phosphate buffer pH 7.4 resulting in the plasmid pMtUMK, which containing 1 mg/ml lysozyme and 1 mM was used for expression of the recom- phenyl methyl sulfonyl fluoride. Cells binant protein fused to His -tag at the 6 were lysed by sonication (6 x 10 s pulses) C-terminus. The whole sequence of pyrH on ice. Clarified cell lysate was obtained in the pMtUMK plasmid was sequenced by centrifugation at 20,800g for 30 min- (Macrogen, Seoul, Korea) to verify the utes at 4ºC. The recombinant protein was absence of any mutation. purified from the supernatant by affinity Overexpression and purification of recom- chromatography using 15 ml TALON® binant MtUMKHis6 Metal affinity column in ÄKTA™ auto- Overexpression of MtUMK in cells mated liquid chromatography system (GE grown in yeast extract tryptone (2xYT) and Healthcare, Uppsala, Sweden) employing in auto-induction medium (ZYP-5052) was a linear gradient of 5 to 400 mM imidazole. 1454 Vol 42 No. 6 November 2011 PURIFICATION OF M. TUBERCULOSIS URIDYLATE KINASE -3 -1 -1 Fractions containing MtUMKHis6 were = 6.22x10 M cm ), compared to the pooled, concentrated and desalted using control in which UMK was absent.