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Cloning and Expression of Bacillus thuringiensis crylAa1 Type Gene.  

이형환 (건국대학교 이과대학 생명과학과)
황성희 (건국대학교 이과대학 생명과학)
권혁한 (건국대학교 이과대학 생명과학)
안준호 (건국대학교 이과대학 생명과학)
김혜연 (건국대학교 이과대학 생명과학)
안성규 (건국대학교 이과대학 생명과학)
박수일 (건국대학교 이과대학 생명과학과)
Publication Information
Microbiology and Biotechnology Letters / v.32, no.2, 2004 , pp. 110-116 More about this Journal
Abstract
The over-expression in E. coli of the pHLN1-SO(+) and pHLN2-80(-) plasmids cloned an insecticidal crystal protein (ICP) gene (crylAal type) from Bacillus thuringiensis var. kurstaki HD 1 was investigated through in part, the deletion of -80 bp promoter and an alternative change of cloning vector system. Two recombinant plasmids were constructed in an attempt to analyze the over-expression of the ICP in relations to its gene structure possessing only -14 bp [Shine-Dalgarno (SD) sequence of -80 bp promoter]. Also, anther two recombinant plasmids similarly cloned the icp gene in a different vector system. The amounts of ICP produced from the recombinants were measured by SDS-PAGE and confirmed by Western blot analysis. One clone, pHLRBS1-14 clone in which only the SD sequence in the inverted orientation icp gene appeared, was more evident than the pHLRBS2-14 clone in which only the -14 bp SD sequence of the right orientated icp gene was shown to exist. The pHLN2-80(-) clone produced more ICP proteins than the pHLRBS1-14 clone. In the two clones, pHLNUC1-80 right-oriented icp gene and the pHLNUC2-80 clone inverted-orientation icp gene in a new different vector, the pHLNUC2-80 produced more ICP proteins in E. coli system. These results indicate that the P/ac promoter, the inverted icp gene insertion and -80 bp promoter (-66 bp part of the icp gene promoters), were concerned with the expression of the icp gene in the recombinant plasmids. In addition, the expression mechanism might result from the disruption of the transcription-suppressing regions in the promoter regions.
Keywords
B. thuringiensis; over-expression; insecticidal protein gene; endotoxin crystal;
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