Co-expression of human malaria parasite Plasmodium falciparum orotate phosphoribosyltransferase and orotidine 5?-monophosphate decarboxylase as enzyme complex in Escherichia coli: a novel strategy for drug development
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Date
2010-07-20
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Asian Biomedicine (Research Reviews and News)
Abstract
Background: Human malaria parasite Plasmodium falciparum operates de novo pyrimidine biosynthetic pathway.
The fifth and sixth enzymes of the pathway form a heterotetrameric complex, containing two molecules each of
orotate phosphoribosyltransferase (OPRT) and orotidine 5’-monophosphate decarboxylase (OMPDC).
Objective: Define the function of OPRT-OMPDC enzyme complex of P. falciparum by co-expressing the enzymes
in Escherichia coli.
Methods: The constructed plasmids containing either P. falciparum OPRT or OMPDC were cloned in E. coli by
co-transformation. Both genes were co-expressed as OPRT-OMPDC enzyme complex and the complex was purified
by chromatographic techniques, including N2+-NTA affinity, Hi Trap Q HP anion-exchange, uridine 5’-
monophosphate affinity, and Superose 12 gel-filtration columns. Physical and kinetic properties of the enzyme
complex were analyzed for its molecular mass.
Results: Co-transformation of PfOPRT and PfOMPDC plasmids in E. coli were achieved with a clone containing
DNA ratio of 1:2, respectively. Both plasmids remained stable and were functionally expressed in the E. coli cell
for at least 20 weeks. The P. falciparum OPRT-OMPDC enzyme complex were co-expressed and the complex was
co-eluted in all chromatographic columns during purification and physical analysis. The molecular mass of the
complex was 130 kDa, whereas the PfOPRT and PfOMPDC component were 35.6 and 41.5 kDa, respectively.
The enzymatic activities of the complex were competitively inhibited by their products of each enzyme
component.
Conclusion: P. falciparum OPRT and OMPDC in E. coli as an enzyme complex were co-transformed and
functionally co-expressed. These have similar properties to the native enzyme purified directly from P.
falciparum, and this character is different from that of the human host organism. The enzyme complex would be
suitable as new target to research selective inhibitors as suitable drugs to better control this disease.
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Keywords
Co-expression, co-transformation, enzyme complex, orotate phosphoribosyltransferase (OPRT), orotidine 5’-monophosphate decarboxylase (OMPDC), Plasmodium falciparum
Citation
Asian Biomedicine (Research Reviews and News); Vol. 4 No. 2 April 2010; 297-306.