Efficient synthesis of mouse thymidylate synthase in Escherichia coli.
The coding region of the mouse thymidylate synthase (TS)-encoding cDNA (ts) was inserted downstream from the phage T7 promoter and translation initiation signals of the expression vector, pET-3a, and transformed into Escherichia coli BL21(DE3)[pLysS]. When the wild-type (wt) cDNA sequence was used, mouse TS was synthesized in the bacterial cells in response to induction, but the level of expression was low. When the second codon (Leu) was changed from CUG, found in the normal mRNA, to CUU, the level of expression increased 17-fold and TS represented 5-10% of total cell protein. The recombinant enzyme was purified to homogeneity by affinity chromatography. The recombinant TS had the same Mr as the enzyme from cultured mouse fibroblasts. Kinetic studies with the recombinant enzyme showed that the apparent Km values for deoxyuridylate and 5,10-methylenetetrahydrofolate were 10.5 and 22 microM, respectively, which were similar to the values for TS from mouse cell extracts. The mouse ts expression vector will be useful for the large-scale production of the wt enzyme and for the creation and analysis of mutant enzymes by protein engineering techniques.
Zhang
H C
HC
Department of Biochemistry, Ohio State University, Columbus 43210.
Cisneros
R J
RJ
Dunlap
R B
RB
Johnson
L F
LF
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D013940:Thymidylate Synthase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D004247:DNA; D013940:Thymidylate Synthase