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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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