title: Heterologous expression of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania major.
abstract: The bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) of Leishmania major has been cloned and expressed in Escherichia coli and Saccharomyces cerevisiae. The strategy involved placing the entire 1560-bp coding sequence into a parent cloning plasmid that was designed to permit introduction of unique restriction sites at the 5'- and 3'-ends. In this manner, the entire coding sequence could be easily subcloned into a variety of expression vectors. High levels of TS-DHFR gene expression were driven by tac, pL and T7 RNA pol promoters in E. coli, and the GAPDH-ADH-2 promoter in S. cerevisiae. L. major TS-DHFR also complemented TS deficiency in E. coli. In E. coli, the protein accumulated to very high levels, but most was present as inactive inclusion bodies. Nevertheless, substantial amounts were soluble; up to 2% of the soluble protein was catalytically active TS-DHFR. In the yeast systems, essentially all of the bifunctional protein was soluble and catalytically active, and crude extracts contained about 100-fold more enzyme than do extracts from wild-type L. major. The expressed TS-DHFR from yeast and E. coli was purified to homogeneity by methotrexate-Sepharose affinity chromatography. About 8.5 mg of homogeneous, catalytically active protein is obtained from a 1-L culture of yeast, and 1.5 mg was obtained from 1 L of E. coli culture. A 200-L fermentation of the yeast expression system yielded a crude extract containing over 4 g of TS-DHFR.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
authors.forename: R
authors.initials: R
authors.lastname: Grumont
authors.forename: W
authors.initials: W
authors.lastname: Sirawaraporn
authors.forename: D V
authors.initials: DV
authors.lastname: Santi
mesh_terms: D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005822:Genetic Vectors; D007895:Leishmania tropica / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D009097:Multienzyme Complexes; C056930:thymidylate synthase-dihydrofolate reductase; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D004262:DNA Restriction Enzymes