Expression of the trimethoprim resistant dihydrofolate reductase encoded by transposon TN4003 in a soluble form and its subsequent purification to homogeneity.
A high level expression in E. coli of the Tmpr type S1 DHFR was achieved by: (1) elimination of an internal start of translation within the RNA, and (2) optimization of gene expression by replacing nucleotides at the 5' end of the gene by nucleotides present in the highly expressible gene for SaDHFR. In addition, by replacing amino acids supposed to be on the surface of the protein, the mutein S1 DHFR[N48E,N130D] was constructed, which can be expressed in E. coli to high levels in a soluble and active form. The mutein S1 DHFR[N48E,N130D] was purified nearly to homogeneity. The enzyme is highly active and remains soluble even at a protein concentration of 10 mg/ml.
Dale
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F. Hoffmann-La Roche Ltd., Pharmaceutical Research-New Technologies, Basel, Switzerland.
Stüber
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Broger
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Langen
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D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004926:Escherichia coli; D015870:Gene Expression; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D014295:Trimethoprim / Q000494:pharmacology*
D004251:DNA Transposable Elements; D011994:Recombinant Proteins; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase