Overproduction of soluble trichodiene synthase from Fusarium sporotrichioides in Escherichia coli.
Trichodiene synthase is a sesquiterpene cyclase isolated from various fungal species which catalyzes the cyclization of farnesyl diphosphate (FPP) to trichodiene. The trichodiene synthase gene (Tox5) of Fusarium sporotrichioides has previously been cloned and expressed as 0.05-0.1% of total cell protein in Escherichia coli. We have used polymerase chain reaction to amplify the trichodiene coding sequence carried on the plasmid pTS56-1. The resulting DNA, carrying a BamHI restriction site and the T7 gene 10 ribosome binding site and translational spacer element immediately upstream of the ATG start codon as well as a HindIII site adjacent to the translational stop codon, was inserted into the corresponding sites of the expression vector pLM1. The latter vector carried the promoter and translational leader sequence from T7 gene 10 and the E. coli rmBT1T2 tandem transcription terminator. This construct was cloned into E. coli BL21 (DE3). The resulting transformants, when induced with isopropyl beta-D-thiogalactoside, produced trichodiene synthase as 20-30% of total soluble protein. The recombinant synthase, which could be purified five-fold to homogeneity by ammonium sulfate precipitation, ion-exchange chromatography on Q Sepharose, and gel filtration on Superose 12, was identical to native protein in steady-state kinetic parameters and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had the expected MENFP N-terminal sequence.
Cane
D E
DE
Department of Chemistry, Brown University, Providence, Rhode Island 02912.
Wu
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Z
Oliver
J S
JS
Hohn
T M
TM
D001483:Base Sequence; D019755:Carbon-Carbon Lyases; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics; D005800:Genes, Fungal; D007700:Kinetics; D008190:Lyases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012717:Sesquiterpenes; D013997:Time Factors
D009838:Oligodeoxyribonucleotides; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D008190:Lyases; D019755:Carbon-Carbon Lyases; C051130:trichodiene synthetase