The Bacillus thuringiensis Cry1Aa toxin:: effects of trypsin and chymotrypsin site mutations on toxicity and stability
The Bacillus thuringiensis Cry1Aa toxin:: effects of trypsin and chymotrypsin site mutations on toxicity and stability
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DOI：
10.1016/j.jip.2004.02.002
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发表时间：
2004-02-01
影响因子：
3.4
通讯作者：
Masson, L
中科院分区：
文献类型：
--
作者：
Bah, A;van Frankenhuyzen, K;Masson, L
本研究的目的是通过突变潜在的凝乳胰蛋白酶和胰蛋白酶位点，创造一种活性Cry1Aa毒素，增强其对云杉budworm （Choristoneura fumiferana）中肠蛋白酶的降解能力。在大肠杆菌-芽孢杆菌穿梭载体中构建了14个Cry1Aa突变体，并在苏云金芽孢杆菌晶体缺失宿主中表达。用云杉芽虫肠液、商品牛胰蛋白酶和糜凝胰蛋白酶对cry1aa野生型和突变型毒素进行了蛋白酶抗性测定。尽管许多突变体几乎没有或没有变化，但在对烟霉菌的生物测定研究中，一些突变体的毒性增加了50倍（R543S， R566G和F570S），毒性增加了50倍（F576S）。体外蛋白酶抗性试验结果表明，其他肠道汁液成分可能参与毒素过度消化。Elsevier Inc.出版。
The objective of the present work was to create an active Cry1Aa toxin showing enhanced resistance to degradation by spruce budworm (Choristoneura fumiferana) midgut proteases by mutating potential chymotrypsin and trypsin sites. Fourteen Cry1Aa mutants were created in an Escherichia coli-Bacillus shuttle vector and expressed in a crystal minus Bacillus thuringiensis host. Using spruce budworm gut juice, commercial bovine trypsin and chymotrypsin we performed protease resistance assays with Cry I Aa wild type and mutant toxins. Although many mutants showed little or no change, several mutants showed a >2-fold increase (R543S, R566G, and F570S) up to a >4-fold increase in toxicity (F576S), in bioassay studies against C fumiferana. The in vitro protease resistance assay results indicated a possible involvement of other gut juice components in toxin overdigestion. Published by Elsevier Inc.