Abstract:
: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.
journal_name
Arch Biochem Biophysjournal_title
Archives of biochemistry and biophysicsauthors
Back K,Yin S,Chappell Jdoi
10.1006/abbi.1994.1533subject
Has Abstractpub_date
1994-12-01 00:00:00pages
527-32issue
2eissn
0003-9861issn
1096-0384pii
S0003986184715335journal_volume
315pub_type
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章,评审
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(87)90099-3
更新日期:1987-04-01 00:00:00
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journal_title:Archives of biochemistry and biophysics
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pub_type: 杂志文章,评审
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
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更新日期:2000-01-15 00:00:00
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journal_title:Archives of biochemistry and biophysics
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更新日期:1996-09-01 00:00:00
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journal_title:Archives of biochemistry and biophysics
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更新日期:2000-12-15 00:00:00