Biotechnology Letters 26: 1051–1055, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.
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Cloning, sequence and expression of the lactate dehydrogenase gene from the human malaria parasite, Plasmodium vivax Dilek Turgut-Balik1,∗ , Ekrem Akbulut1, Debbie K. Shoemark2 , Venhar Celik1 , Kathleen M. Moreton2 , Richard B. Sessions2 , J. John Holbrook2,† & R. Leo Brady2 1 University
of Firat, Faculty of Arts and Sciences, Department of Biology 23169, Elazig, Turkey Biochemistry, University of Bristol, University Walk Bristol, BS8 1TD, UK ∗ Author for correspondence (Fax: +0090 4242330062; E-mail:
[email protected]) 2 Department of
Received 30 March 2004; Revisions requested 6 April 2004; Revisions received 22 April 2004; Accepted 24 April 2004
Key words: anti-malarial, gene cloning, homology model, lactate dehydrogenase, Plasmodium vivax
Abstract Increased drug resistance to anti-malarials highlights the need for the development of new therapeutics for the treatment of malaria. To this end, the lactate dehydrogenase (LDH) gene was cloned and sequenced from genomic DNA of Plasmodium vivax (PvLDH) Belem strain. The 316 amino acid protein-coding region of the PvLDH gene was inserted into the prokaryotic expression vector pKK223-3 and a 34 kDa protein with LDH activity was expressed in E. coli. Structural differences between human LDHs and Pf LDH make the latter an attractive target for inhibitors leading to novel anti-malarial drugs. The sequence similarity between PvLDH and Pf LDH (90% residue identity and no insertions or deletions) indicate that the same approach could be applied to Plasmodium vivax, the most common human malaria parasite in the world.
Introduction Malaria is one of the most widespread infectious diseases affecting some 500 million people with an enormous cost in human suffering and economic hardship. Effective treatment of the disease is increasingly compromized by rising resistance of malaria parasites to currently available anti-malarials. The parasites are homolactate fermenters and rely on glycolysis for energy generation (Sherman 1979) since the parasites appear to lack a functional citric acid cycle. The NAD+ consumed during glycolysis is reduced to NADH by lactate which, in turn, is oxidized to pyruvate. This reaction is catalyzed by lactate dehydrogenase (LDH). It has been demonstrated that inhibitors of this enzyme have parasiticidal activity (Royer et al. 1986, Prof S. Croft, London School of Hygiene and Tropical Medicine, pers. commun.). Since LDH from the malaria parasite Plasmodium falciparum (Pf LDH) has notable structural and kinetic differences (Dunn et al. † Deceased.
1996, Gomez et al. 1997) from human LDHs, the enzyme appears to be an attractive target for novel anti-malarial therapeutics (Sherman 1979, Roth et al. 1988, Oelshlegel et al. 1975, Dunn et al. 1996, Gomez et al. 1997, Nwaka & Ridley 2003). The parasite, P. falciparum, is the most lethal of malarial plasmodia being responsible for the cerebral form of the disease, consequently it has been the major focus of initial biochemical and genomic investigation (Bzik et al. 1993, Bernal et al. 2001, Turgut-Balik & Holbrook 2001). On the other hand, the parasite P. vivax is of great importance as it is the most widespread and common of the malarial plasmodia and, therefore, is responsible for the greatest burden of disease. In this paper we report the cloning and sequencing of the LDH gene from P. vivax and show that the expression product of the gene in E. coli is a protein of the anticipated size and lactate dehydrogenase activity. This opens the door to the development of structure-based inhibitors to this target, either separate from, or in conjunction with such work being
1052 performed with Pf LDH with the longer term aim of developing anti-malarials active against both species of the disease.
transformed into using CaCl2 competent (Sambrook & Russell 2001) E. coli JM101 cells. Both ligation and transformation were performed according to the suppliers’ instructions.
Materials and methods
DNA sequencing
Plasmodium vivax genomic DNA
Plasmid inserts were checked by colony PCR using the Pv3 and Pv4 oligonucleotide primers and a product of the correct size were obtained. Plasmid DNA was prepared using Wizard Plus SV Minipreps DNA Purification System (Promega, UK) and submitted for sequencing from both directions.
Genomic DNA was obtained from two different sources. Firstly, genomic DNA from the Belem strain was kindly provided by Dr P. Davis (Pasteur Institute, Paris, France). Secondly, P. vivax infected blood samples were obtained from The Diyarbakir Malaria and Tropical Diseases Research and Education Center Malaria Unite, Diyarbakir, Turkey in the form of air-dried spots on 3 MM Whatman filter paper (25 µl P. vivax infected blood samples per spot). Genomic DNA was isolated from these blood spots according to the procedure described by Kain et al. (1993). Amplification of the P. vivax LDH gene by PCR Two oligonucleotide primers were prepared, complementary to the forward and reverse strands of the PvLDH (lactate dehydrogenase) gene based on the putative sequence of PvLDH recently deposited in the database of the TIGR P. vivax Genome Project. The forward primer was Pv3, 5 ATGACGCCGAAACCCAAAATTGTGCTCGTCGGG-3 , and the reverse primer was Pv4, 5 -AATGAGCGCCTTCATCCTTTTAGTCTCCGC-3 . The reaction mixture used for gene amplification contained 5 µl AmpliTaq Gold DNA Polymerase buffer II, which is supplied with the enzyme (Applied Biosystems, USA), 1.5 mM MgCl2 , 5 µl stock dNTPs (10 µl each 10 mM dNTPs and 10 µl H2 O). 50 pmol of each oligonucleotide primer, 1 µl genomic DNA from P. vivax Belem strain (stock 0.5 µg µl−1 ), 0.5 µl (2.5 units) of Ampli Taq Gold DNA Polymerase and water to a final volume of 50 µl. PCR conditions were: denaturation at 94 ◦ C for 1.5 min, annealing at 55 ◦ C for 2 min and extension at 72 ◦ C for 2 min for 45 cycles. Electrophoresis of the PCR product on a 1% agarose gel gave a DNA band of the expected size which was purified using Promega’s Wizard SV Gel and PCR Clean-Up System. Ligation and transformation The PCR product was ligated into plasmid DNA using the pGEM-T easy vector system (Promega, UK) and
Expression and LDH-activity of PvLDH The Pv3 and Pv4 primers were altered to assist ligation into the expression vector pKK223-3 such that the forward primer included an EcoRI cleavage site, and the reverse primer had a PstI cleavage site. The plasmid containing the PvLDH gene was transformed into CaCl2 competent DH5α E. coli cells. A starter-culture was grown overnight at 37 ◦ C in double strength yeast tryptone (2 × YT) broth, supplemented with 100 µg ampicillin ml−1 . Further 50 ml samples of the growth medium were inoculated with 500 µl of the starter culture grown to OD600 0.6 at 37 ◦ C. Sample growth was continued overnight both with and without induction using 1 mM IPTG. Aliquots (3 ml) were taken and the cells harvested by centrifugation in a microfuge for 1 min. The cell supernatant was removed and the cell pellet was resuspended in 500 µl 50 mM TEA, pH 7.5. Cell suspensions were disintegrated ultrasonically in ice and centrifuged at 4 ◦ C for 5 min in a microfuge. The protein content of the supernatant and pellet fractions was visualized by SDS-PAGE. The supernatants were also analyzed for relative LDH activity by following the loss of NADH (A at 340 nm) in the presence of pyruvate under standard conditions (Turgut-Balık et al. 2001).
Results and discussion Amplification, cloning and DNA sequencing of the P. vivax LDH gene Before the availability of P. vivax genomic data, degenerate primers based on the Pf LDH gene sequence were used in attempts to amplify the PvLDH gene from P. vivax infected blood samples and this procedure gave a 273 bp product. The coding sequence of this
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Fig. 1. Amino acid sequence alignment (DnaStar) of PvLDH to human and plasmodia LDHs. Key catalytic residues are underlined and residues differing between PvLDH and Pf LDH are in bold.
PCR product showed high amino-acid identities with other plasmodial LDHs including the presence of key catalytic residues (data not shown). Once the putative PvLDH gene sequence was published in the TIGR database of the P. vivax Genome sequencing Project, specific primers were designed as described in Materials and methods. These primers allowed amplification of the full-length PvLDH gene from from P. vivax Belem genomic DNA. This PCR product was cloned into the ‘pGEM-T Easy Vector’ plasmid and sequenced from both directions. Sequence comparison with other LDHs confirmed the presence of the PvLDH gene in the vector.
the key catalytic residues in these enzymes are completely conserved across species, including: arginine171 (binds pyruvate), aspartate-168 and histidine-195, (the proton donor couple) and arginine-109 (polarizes the pyruvate carbonyl group) leaving little doubt that the cloned gene is PvLDH. The 31 amino acid differences between PvLDH and Pf LDH were modelled onto the crystal structure of Pf LDH and these changes are largely distributed across the surface of the protein, remote from the active site (see Figure 2). High confidence in such a model arises from the 90% sequence identity between PvLDH and PbLDH and the fact that the crystal structures of Pf LDH and PbLDH (92% sequence identity) are similar (Winter et al. 2003).
Sequence comparison of PvLDH with known LDHs Overproduction and enzymic activity of PvLDH Figure 1 shows a sequence alignment of PvLDH with known plasmodial LDHs and the major human isoforms. The amino acid sequence of PvLDH shows no inserts of deletions with respect to the other plasmodial LDHs and a high sequence identity (90% identical with each of Pf LDH, P. berghei LDH (PbLDH), and P. yoelii LDH (PyLDH)). The level of amino acid identity with the two human LDHs, human-M4 and human-H4 is correspondingly low, 26% and 25% respectively. Nonetheless, all of
E. coli cells transformed with the plasmid containing the PvLDH gene were grown overnight both with and without induction, as described in Materials and methods, and compared with similar growths of untransformed cells. Analysis of the cell pellets by SDSPAGE revealed over-production of a protein with a molecular weight around 34 kDa in both the induced and non-induced cells (data not shown). This protein remained in the supernatant fraction after sonication
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Fig. 2. A homology model of PvLDH based on the crystal structure of Pf LDH. The backbone is shown as a ribbon and the side-chains of residues that differ between PvLDH and Pf LDH are represented as ball & sticks. The five-residue insertion in the active-site loop, which is conserved in plasmodia compared with all other LDHs, is shown in green.
and centrifugation of the cell pellet. These supernatant fractions were assayed for LDH activity using pyruvate as substrate. Both induced and non-induced samples gave a >10 fold increase in LDH activity compared with endogenous E. coli LDH activity from the untransformed cells (Table 1). Hence the transformed cells produce soluble and active PvLDH. The promoter region on the pKK223-3 expression vector is not tightly controlled by IPTG such that protein is constitutively expressed at low levels. Under the conditions described here, induction with IPTG did not increase the yield of active PvLDH.
Conclusion The cloning of the LDH gene from P. vivax and the over-production of the LDH protein gene product as a
Table 1. Comparative lactate dehydrogenase (LDH) activities measured in Enzyme Units (EU) per ml cells cultured overnight, normalized to an optical density (OD) of 1 at 600 nm. For LDH an EU is the amount of enzyme required to turn-over 1 µmol of NADH per min. E. coli DH5α
EU per ml cells
Untransformed Transformed Transformed & induced
0.025 0.47 0.38
soluble, active enzyme is described here. This work opens up the route to further structural and kinetic studies towards development of inhibitors and hence therapeutic agents against this most common species of the malaria parasite, Plasmodium vivax.
1055 Acknowledgements We thank Prof P. Davis, Pasteur Institute, Paris, France, for providing Plasmodium vivax genomic DNA (Belem strain), and the members of the Diyarbakir Malaria and Tropical Diseases Research and Education Center Malaria Unite, Diyarbakir, Turkey, for providing Plasmodium vivax infected blood samples. This project was supported by: The Wellcome Trust (Grant No: 060406 UK), TUBITAK (Project No: TBAG 1969) and Firat University Scientific Research Unit (Project No: 565, 682, 694).
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