Cloning and Expression in Escherichia coli of the 135-kDa Insecticidal Protein Gene from Bacillus thuringiensis subsp. aizawai IPL7

抄録

Bacillus thuringiensis subsp. aizawai IPL7 produced 130-kDa and 135-kDa insecticidal proteins. The 135-kDa protein gene was cloned from a cured derivative of strain IPL7, which lacked the 130-kDa protein gene. The cloned 3.6-kb DNA fragment contained a 3, 623-bp protein-coding region with 72-bp 5'-flanking and 20-bp 3'-flanking regions. The open reading frame encoded a 133, 161-Da protein consisting of 1, 176 amino acid residues. When compared with the amino acid sequence of the 130-kDa protein reported previously, it was found that amino acid substitutions mainly occurred in three regions; amino acid residues 206 to 455, 788 to 819, and 1, 061 to 1, 137 of the 135-kDa protein. The cloned 135-kDa protein gene was expressed in Escherichia coli under the control of the tac promoter and rrnB terminator. The synthesized 135-kDa protein amounted to 30% of the total cellular protein. The purified 135-kDa protein had insecticidal activity against Plutella xylostella larvae.