Expression of a plant sesquiterpene cyclase gene in Escherichia coli.
5-Epi-aristolochene synthase is a sesquiterpene cyclase activity found in pathogen-challenged tobacco cells, but not in nonchallenged tissues, and appears to be encoded by a complex gene family. As a prerequisite to assessing the functional significance of these multiple genes, bacterial expression systems were examined for their capacity to express a tobacco sesquiterpene cyclase cDNA. Insertion of full-length 5-epi-aristolochene synthase cDNA into two commonly used expression vectors, pET-11d and pGBT-T19, resulted in high level expression of the cyclase activity. The highest level of expression occurred 3 h after induction with low concentrations (0.1-0.5 mM) of IPTG, incubation at 27 degrees C instead of 37 degrees C, and in the bacterial host strain BL21(DE3). Under these conditions, the cyclase protein constituted 5 to 8% of the soluble and 35% of the total Escherichia coli proteins. Enzyme reaction products of the native tobacco and recombinant enzyme were identical, based on argentation-thin layer chromatography. Deletion mutants of the cyclase gene corresponding to the amino and carboxy termini of the enzyme were prepared. The cyclase proteins resulting from bacterial expression of these mutant constructs were found largely in the insoluble protein fraction and no soluble enzyme activity was detected.
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Agronomy Department, University of Kentucky, Lexington 40546-0091.
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Chappell
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D019883:Alkyl and Aryl Transferases; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli; D015870:Gene Expression; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D013329:Structure-Activity Relationship; D013662:Tea / Q000235:genetics; D013696:Temperature; D014026:Nicotiana / Q000235:genetics*; D014166:Transferases / Q000235:genetics*
D017931:DNA Primers; D013662:Tea; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C078217:5-epi-aristolochene synthase