Worachart SirawarapornRachada SirawarapornYongyuth YuthavongAlan F. CowmanDaniel V. SantiMahidol UniversityWalter and Eliza Hall Institute of Medical ResearchUniversity of California, San Francisco2018-06-142018-06-141990-12-01Biochemistry. Vol.29, No.48 (1990), 10779-1078515204995000629602-s2.0-0025605441https://repository.li.mahidol.ac.th/handle/123456789/15911The coding sequence of the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from a moderately pyrimethamine-resistant strain (HB3) of Plasmodium falciparum was assembled in a pUC expression vector. The coding sequence possesses unique Nco 1 and Xba1 sites which flank 243 bp of the DHFR gene that include all point mutations thus far linked to pyrimethamine resistance. Wild-type (3D7) and highly pyrimethamine-resistant (7G8) TS-DHFRs were made from this vector by cassette mutagenesis using Nco1-Xba1 fragments from the corresponding cloned TS-DHFR genes. Catalytically active recombinant TS-DHFRs were expressed in Escherichia coli, albeit at low levels. Both TS and DHFR coeluted upon gel filtration and copurified upon affinity and anion exchange chromatography. Gel filtration and SDS-PAGE indicated that the enzyme was a dimer with identical 67-kDa subunits, characteristic of protozoan TS-DHFRs. Amino-terminal sequencing gave 10 amino acids which perfectly matched the sequence predicted from the nucleotide sequence. The recombinant TS-DHFR was purified to homogeneity by 10-formylfolate affinity chromatography followed by Mono Q FPLC. The inhibition properties of pyrimethamine toward the purified recombinant enzymes show that the point mutations are the molecular basis of pyrimethamine resistance in P. falciparum. © 1990, American Chemical Society. All rights reserved.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyArticleSCOPUS10.1021/bi00500a009