Tan Y, Donovan W P
Ecogen, Inc., 2000 Cabot Boulevard West, Langhorne, Pennsylvania, PA 19047, USA.
J Biotechnol. 2001 Nov 17;84(1):67-72. doi: 10.1016/s0168-1656(00)00328-x.
The aprA gene encoding alkaline protease A (AprA) was cloned from Bacillus thuringiensis subsp. kurstaki, and the cloned gene was used to construct aprA-deleted (aprA1) strains of B. thuringiensis. An aprA1 strain of B. thuringiensis that contained the wild-type gene for neutral protease A (nprA(+)) displayed levels of extracellular proteolytic activity that were similar to those of an aprA(+)nprA(+) strain. However, when EDTA was included in the protease assay to inhibit NprA activity the aprA1nprA(+) strain displayed only 2% of the extracellular proteolytic activity of the aprA(+)nprA(+) strain. A strain that was deleted for both aprA and nprA (aprA1nprA3 strain) failed to produce detectable levels of proteolytic activity either in the presence or absence of EDTA in the assay. Compared with the aprA(+)nprA(+) strain the aprA1nprA(+) strain yielded 10% more full-length Cry1Bb crystal protein and the aprA1nprA3 strain yielded 25% more full-length Cry1Bb protein. No significant differences were seen in the 50% lethal dose of Cry1Bb protein from aprA(+)nprA(+) and aprA1nprA3 strains against three species of lepidopteran insects. These results suggest that enhanced yield of certain crystal proteins can be obtained by deletion of the genes aprA and nprA which are the major extracellular proteases of B. thuringiensis.
从苏云金芽孢杆菌库斯塔克亚种中克隆了编码碱性蛋白酶A（AprA）的aprA基因，并使用该克隆基因构建了苏云金芽孢杆菌的aprA缺失（aprA1）菌株。含有中性蛋白酶A野生型基因（nprA(+)）的苏云金芽孢杆菌aprA1菌株表现出的胞外蛋白水解活性水平与aprA(+)nprA(+)菌株相似。然而，当在蛋白酶测定中加入EDTA以抑制NprA活性时，aprA1nprA(+)菌株的胞外蛋白水解活性仅为aprA(+)nprA(+)菌株的2%。在测定中无论有无EDTA，同时缺失aprA和nprA的菌株（aprA1nprA3菌株）都未能产生可检测水平的蛋白水解活性。与aprA(+)nprA(+)菌株相比，aprA1nprA(+)菌株产生的全长Cry1Bb晶体蛋白多10%，aprA1nprA3菌株产生的全长Cry1Bb蛋白多25%。aprA(+)nprA(+)和aprA1nprA3菌株的Cry1Bb蛋白对三种鳞翅目昆虫的50%致死剂量没有显著差异。这些结果表明，通过缺失苏云金芽孢杆菌的主要胞外蛋白酶基因aprA和nprA，可以提高某些晶体蛋白的产量。
