Expression of human thymidylate synthase in Escherichia coli.
A cDNA clone encoding thymidylate synthase (TS) has been isolated from a human T-cell library and modified in the 5'-untranslated region to incorporate several unique cloning sites. The gene has been cloned as a cassette into several Escherichia coli expression vectors which did not provide detectable amounts of the enzyme. A successful approach used a constitutive E. coli expression vector developed for the enzyme from Lactobacillus casei. A 115-base pair 5'-untranslated region from the L. casei TS which contains a ribosomal binding site and other regulatory sequences has been fused to the coding region of the human TS gene to provide a construct that is expressed in E. coli. The level of expression was further enhanced by altering the nucleotide sequence of the first 90 base pairs to accommodate common codon use in E. coli. In our best expression system, catalytically active human TS is expressed to a level that represents about 1.6% of the total soluble protein. The recombinant human TS has been purified and characterized; except for the presence of an amino-terminal blocking group, the enzyme has physical and kinetic properties similar to the enzyme isolated from human cells.
Davisson
V J
VJ
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Sirawaraporn
W
W
Santi
D V
DV
D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D013329:Structure-Activity Relationship; D013940:Thymidylate Synthase / Q000235:genetics* / Q000302:isolation & purification
D004269:DNA, Bacterial; D004247:DNA; D013940:Thymidylate Synthase