Expression and Enzyme Kinetics of Fused Plasmodium falciparum Orotate Phosphoribosyltransferase and Orotidine 5′-monophosphate Decarboxylase in Different Escherichia Coli

Waranya Imprasittichai1, *, Sudaratana R. Krungkrai2, Jerapan Krungkrai3, Patsarawadee Paojinda4
1 Department of Basic Medical Science, Faculty of Medicine Vajira Hospital, Navamindradhiraj University, Bangkok 10300, Thailand
2 Unit of Biochemistry, Department of Medical Science, Faculty of Science, Rangsit University, Patumthani 12000, Thailand
3 Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
4 Department of Medical Technology, Faculty of Science and Technology, Bansomdejchaopraya Rajabhat University, Bangkok 10600, Thailand

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© 2023 Imprasittichai et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Department of Basic Medical Science, Faculty of Medicine Vajira Hospital, Navamindradhiraj University, Bangkok 10300, Thailand;



Fusion of the last two enzymes in the pyrimidine biosynthetic pathway in the inverse order by having COOH-terminal orotate phosphoribosyltransferase (OPRT) and NH2-terminal orotidine 5′-monophosphate decarboxylase (OMPDC), as OPRT-OMPDC, has been described in many organisms.


The study aimed to select the optimum host cell and temperature for expressing the recombinant fusion OMPDC-OPRT having the enzymatic activity.


We constructed gene fusions of the human malaria parasite Plasmodium falciparum OMPDC-OPRT (1,836 bp) in the pTrcHisA vector and expressed it as a 6xHis-tag bifunctional protein in three Escherichia coli strains (BL21(DE3), TOP10, Rosetta) at 18°C and 25°C. The recombinant bifunctional protein was partially purified by Ni-nitrilotriacetic acid affinity chromatography and confirmed via Western blot and LC-MS/MS. The enzyme kinetics of OPRT and OMPDC was assessed.


Specific enzymatic activities of both OPRT and OMPDC domains expressed in E. coli BL21(DE3) cells were approximately eight-to-nine-fold higher than those in the TOP10 cells at 18°C. However, the specific activities of both domains expressed in the TOP10 cells were twice higher than those of the BL21(DE3) cells at 25°C. Very low and no enzymatic activities were observed when the constructed vector was expressed in the Rosetta cells at both induction temperatures. The bifunctional enzyme had specific activities of the OPRT and OMPDC domains in a ratio of 1:2. Kinetic study values of the OPRT domain in the bifunctional OMPDC-OPRT enzyme were found to be relatively low at µM level and at the perfect catalytic efficiency (kcat/Km).


The recombinant fusion of OMPDC-OPRT exhibited a high expression level of E. coli BL21(DE3) at 18°C. The kinetic parameter is greater than 108M-1s-1.

Keywords: Plasmodium falciparum, Bifunctional enzyme, Orotate phosphoribosyltransferase, Orotidine 5′-monophosphate decarboxylase, Expression, TOP10 cells.