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Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/15911
Title: Heterologous Expression of Active Thymidylate Synthase–Dihydrofolate Reductase from Plasmodium falciparum
Authors: Worachart Sirawaraporn
Rachada Sirawaraporn
Yongyuth Yuthavong
Alan F. Cowman
Daniel V. Santi
Mahidol University
Walter and Eliza Hall Institute of Medical Research
University of California, San Francisco
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 1-Dec-1990
Citation: Biochemistry. Vol.29, No.48 (1990), 10779-10785
Abstract: The 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.
URI: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=0025605441&origin=inward
http://repository.li.mahidol.ac.th/dspace/handle/123456789/15911
ISSN: 15204995
00062960
Appears in Collections:Scopus 1969-1990

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