High-level expression of human thymidylate synthase.
A method is presented for expressing human thymidylate synthase (TS) to the extent of 25-30% of the protein in Escherichia coli. By this procedure, 200-400 mg of pure enzyme can be obtained from a 2-liter culture of cells. The key to the level of expression appears to be related to the conversion of purine bases in the third, fourth, and fifth codons of the TS cDNA to thymine, without altering the encoded protein product. Conversion of the penultimate proline to a leucine did not diminish expression, but while the isolated native enzyme contained only proline on its amino-terminal end, the mutated enzyme was found to contain methionine on its amino terminus. By contrast, the expression of the unmodified TS cDNA represented only about 0.1-0.2% of the total cellular protein. Unlike recombinant rat and human TSs, the respective enzymes purified to homogeneity from eukaryotic cells were blocked at the amino ends and possessed 2- to 4-fold lower specific activities. To determine at what level the impairment of expression occurred, an in vitro transcription, translation system was employed and the results showed that while transcription was unaffected, the translation of native TS mRNA was reduced by at least 20-fold relative to modified TS mRNA using a rabbit reticulocyte translation system. Thus, it appears that at least for the TS gene, expression is greatly influenced by the GC content of the 5' coding region of the gene in both prokaryote and eukaryote systems.
Pedersen-Lane
J
J
New York State Department of Health, Wadsworth Center, Albany 12201, USA.
Maley
G F
GF
Chu
E
E
Maley
F
F
D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013940:Thymidylate Synthase / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D013940:Thymidylate Synthase