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

Background: Fusion of the last two enzymes in pyrimidine biosynthetic pathway inverse order by having COOH-terminal orotate phosphoribosyltransferase (OPRT) and NH 2 -terminal orotidine 5′-monophosphate decarboxylase (OMPDC), as OPRT-OMPDC, has been described many organisms. Objective: The study aimed to select optimum host cell temperature for expressing recombinant fusion OMPDC-OPRT enzymatic activity. Methods: We constructed gene fusions human malaria parasite Plasmodium falciparum (1,836 bp) pTrcHisA vector expressed it a 6xHis-tag bifunctional protein three Escherichia coli strains (BL21(DE3), TOP10, Rosetta) at 18°C 25°C. was partially purified Ni-nitrilotriacetic acid affinity chromatography confirmed via Western blot LC-MS/MS. enzyme kinetics OPRT OMPDC assessed. Results: Specific activities both domains E. BL21(DE3) cells were approximately eight-to-nine-fold higher than those TOP10 18°C. However, specific twice Very low no observed when Rosetta induction temperatures. had ratio 1:2. Kinetic values domain found be relatively µM level perfect catalytic efficiency ( k cat /K m ). Conclusion: exhibited high expression kinetic parameter is greater 10 8 M -1 s .