Nucleotide sequence of the insecticidal protein gene of Bacillus thuringiensis strain aizawai IPL7 and its high-level expression in Escherichia coli |
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| Authors: | K Oeda K Oshie M Shimizu K Nakamura H Yamamoto I Nakayama H Ohkawa |
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| Institution: | 1. Biotechnology Laboratory, Takarazuka Research Center, Sumitomo Chemical Co., Ltd., Takarazuka, Hyogo 665 Japan (0797)73-0221;2. Pesticide Laboratory, Takarazuka Research Center, Sumitomo Chemical Co., Ltd., Takarazuka, Hyogo 665 Japan (0797)73-0221;1. National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand;2. School of Bioresources and Technology, King Mongkut''s University of Technology Thonburi, Bangkok 10140, Thailand;1. National Center for Geriatrics and Gerontology, Obu, Aichi, 474-8511, Japan;2. Department of Advanced Medical Science, Asahikawa Medical University, Japan;1. Department of Medicine, Rush University Medical Center, Chicago, IL 60612, USA;2. Department of Immunology/Microbiology, Rush University Medical Center, Chicago, IL 60612, USA;3. Cancer Center, Rush University Medical Center, Chicago, IL 60612, USA;1. Department of Physiology and Pharmacology, Federal University of Ceará, Fortaleza, Ceará, Brazil;2. Department of Clinical and Toxicological Analyses, Federal University of Ceará, Fortaleza, Ceará, Brazil;3. Institute of Biosciences of Rio Claro, São Paulo State University, Brazil;4. Computer Engineering, Federal University of Ceará, Sobral, Ceará, Brazil;5. Department of Biophysics and Pharmacology, Federal University of Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil;6. Department of Physical, Federal University of Ceará, Fortaleza, Ceará, Brazil |
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| Abstract: | A DNA fragment carrying the insecticidal protein gene of Bacillus thuringiensis subsp. aizawai IPL7 was cloned from a 78-kb plasmid. The nucleotide sequence revealed that the cloned DNA fragment contained a 3465-bp protein-coding region with 156-bp 5'-flanking, and 168-bp 3'-flanking regions. The open reading frame encoded a 130,690 Da protein consisting of 1155 amino acid residues. Nucleotide sequence comparison of the aizawai gene with the published berliner 1715 gene showed only 8 nt changes in the coding regions. It was found that 72 bp of the 5'-flanking sequence of the cloned aizawai gene was responsible for constitutive expression of the 130-kDa protein gene in Escherichia coli. The expression was greatly enhanced by introducing the tac promoter upstream from the 72-bp 5'-flanking region of the aizawai gene. Under optimal conditions, the 130-kDa insecticidal protein amounted to 38% of the total cellular protein. |
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