Cloning and Expression of Candidate Drug Target Protien from Plasmodium Falciparum in Escherichia coli

  • Woranop Sukparangsi

  • Dr.Pongchai Harnyuttanakorn


Chulalongkorn University






01 มกราคม 2541


An enzyme, deoxyuridine 5’ triphosphate nucleotidohydrolase (dUTPase), catalyses the hydrolysis of dUTP to yield dUTP and inorganic pyrophosphate. The reaction is important because it generates the thymidine biosynthesis precursor dUMP and maintains low concentration of dUTP preventing this nucleotide to be incorporated into DNA. The enzyme in encoded by a dutpase gene. The project goal was to study this gene by cloning and protein expression to collect data for future use the enzyme as an effective drug target. Plasmodium falciparum genomic DNA extracted by phenol chloroform extraction. The genomic DNA had ![OD_{260/280}](/latexrender/pictures/6a4/6a431a3cefb2e328af94003b88fd25db.gif)approximate 2.48, and its concentration was 500 ng/ml. Using primers designed for dUTPase gene, Left primer and right primer had size 55 bases and 57 bases, and melting point is 55.84 and 59.39, respectively. Optimization of the PCR condition illustrated that the optimal genomic DNA concentration for PCR was 1:100 and optimal annealing temperature was ![62^0](/latexrender/pictures/6a4/228/2289bbeb8bb2ab681754da8d61faee05.gif). PCR product was synthesized and purified by QIAquick PCR purification kit. In order to transform the target gene into bacteria cells using a vector called pGEX 2TM(Sittiporn, 2004) extracted by plasmid minipreparation. Both DNA molecule were digested by enzyme BamHl and Kpnl and were ligated by T4 ligase to form recombinant plasmid. Consequently, Plasmids were transformed into E.coli competent cell using ![CaCl_2](/latexrender/pictures/6a4/228/bdf/bdfd297aaa16972580116b8aff992d75.gif) method.