Frontier Research

New Expression System for DHFR-TS protein from P. vivax for antifolate screening application

Walailak Frontier 14, March 2019

 

New Expression System for DHFR-TS protein from P. vivax for antifolate screening application

 

Plasmodium vivax is still the most prevalent cause of human malaria in Asia and South and Central America. One promising drug target for the treatment of P. vivax is dihydrofolate reductase-thymidylate synthase (DHFR-TS), a key enzyme in folate biosynthesis and utilization. Unlike the case for P. falciparum, drug susceptibility test for P. vivax is difficult because of the lack of a continuous in vitro culture for this parasite. To facilitate antifolate screening for P. vivax, we have generated an in vivo model of P. berghei expressing P. vivax DHFR-TS (PvDHFR-TS) of wild-type (WT) or double mutant (SP21) enzymes.

Plasmid construction bearing pvdhfr-ts (WT) gene with homologous sequences for Plasmodium berghei transfection

 

The healthy schizont obtained from overnight in vitro culture followed by density gradient separation was used for P. berghei transfection. P. berghei was transfected with constructed plasmids bearing pvdhfr-ts (WT) and mutant (SP21) alleles. Double crossover homologous recombination was used to replace the endogenous pbdhfr-ts with P. vivax homologous genes. The integration was verified by Southern analysis and transgenic P. berghei cloning was performed using FACS sorting. The transgenic parasites lines validated as models by standard drug screening assays.
 
Plasmid construction bearing pvdhfr-ts (SP21) with homologous sequence for Plasmodium berghei transfection, and validated transgenic Plasmodium berghei lines for antimalarial drug screening
 
With the permanent integration of pvdhfr-ts gene in the genome, the transgenic P. berghei lines expressing PvDHFR-TS are genetically stable and will be useful for antifolate screening targeting PvDHFR-TS. A similar approach could be used to generate transgenic models specific for other targets of interest, thus facilitating the development of anti-P. vivax drugs in general.

 

Sources:

  • Somsak V, Uthaipibull C, Prommana P, Srichairatanakool S, Yuthavong Y, Kamchonwongpaisan S. Transgenic Plasmodium parasites stably expressing Plasmodium vivax dihydrofolate reductase-thymidylate synthase as in vitro and in vivo models for antifolate screening. Malaria Journal. 2011; 10:291
  • Somsak V, Srichairatanakool S, Kamchonwongpaisan S, Yuthavong Y, Uthaipibull C. Small-scale in vitro culture and purification of Plasmodium berghei for transfection experiment. Molecular and Biochemical Parasitology. 2011; 177(2):156-9

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