Cloning and heterologous expression of Plasmodium ovale dihydrofolate reductase-thymidylate synthase gene

https://doi.org/10.1016/j.parint.2011.12.004Get rights and content
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Abstract

Plasmodial bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a validated antimalarial drug target. In this study, expression of the putative dhfr-ts of Plasmodium ovale rescued the DHFR chemical knockout and a TS null bacterial strain, demonstrating its DHFR and TS catalytic functions. PoDHFR-TS was expressed in Escherichia coli BL21 (DE3) and affinity purified by Methotrexate Sepharose column. Biochemical and enzyme kinetics characterizations indicated that PoDHFR-TS is similar to other plasmodial enzymes, albeit with lower catalytic activity but better tolerance of acidic pH. Importantly, the PoDHFR from Thai isolate EU266602 remains sensitive to the antimalarials pyrimethamine and cycloguanil, in contrast to P. falciparum and P. vivax isolates where resistance to these drugs is widespread.

Graphical abstract

Highlights

► Polymorphism of P. ovale dihydrofolate reductase-thymidylate synthase was identified. ► Bacterial complementation assay revealed the function of the putative PoDHFR-TS. ► The protein was expressed and purified. ► Biochemical and kinetic properties including antifolate sensitivity were determined.

Abbreviations

DHFR
dihydrofolate reductase
TS
thymidylate synthase
JR
junction region
Pf
Plasmodium falciparum
Po
P. ovale
Pv
P. vivax
Tc
Trypanosoma cruzi
CYC
cycloguanil
DTT
dithiothreitol
dUMP
deoxyuridine monophosphate
EDTA
ethylenediaminetetraacetic acid
HCOH
formaldehyde
H2folate
7,8-dihydrofolate
IPTG
iso-propyl-beta-d-thio-galactopyranoside
LC-MS/MS
liquid chromatography-mass spectrometry
MTX
methotrexate
NADPH
nicotinamide adenine dinucleotide phosphate
PVDF
polyvinylidene fluoride
PYR
pyrimethamine
TES
N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid
TMP
trimethoprim
6-[3H] FdUMP
5-fluoro 2′-deoxyuridine 5′-monophosphate
(6R)-CH2H4folate
5,10-methylene tetrahydrofolate
MM
minimum media

Keywords

Plasmodium ovale
Dihydrofolate reductase-thymidylate synthase
Antifolates

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