Functional expression of the dihydrofolate reductase and thymidylate synthetase activities of the human malaria parasite Plasmodium falciparum in Escherichia coli.
We have developed a recombinant system that directs the functional expression from Escherichia coli of both dihydrofolate reductase-thymidylate synthetase (DHFR-TS) and the isolated DHFR domain from Plasmodium falciparum. Both products are inhibitory to a number of E. coli cell lines to the extent that cell growth ceases immediately upon induction. This dramatic inhibition is not seen in strain AB1899, in which amounts of plasmodial protein of up to 100 times the basal E. coli TS level can be accumulated. However, as well as the full-length DHFR-TS molecule, smaller proteins carrying an intact TS substrate-binding site are produced. These represent ca. 60-75% of the total plasmodial protein expressed and are observed in every E. coli strain examined. We show that they are not derived by degradation of the parent DHFR-TS molecule, but can be correlated with the sizes of proteins expected to be produced if erroneous initiation of translation were occurring at 3 internal methionine residues.
Hall
S J
SJ
Department of Biochemistry and Applied Molecular Biology, University of Manchester Institute of Science and Technology, U.K.
Sims
P F
PF
Hyde
J E
JE
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D010957:Plasmids; D010963:Plasmodium falciparum / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*; D013940:Thymidylate Synthase / Q000096:biosynthesis / Q000235:genetics*; D014296:Trimethoprim Resistance / Q000235:genetics
D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase