Abdullah Mohd Amir F, Alzate Oscar, Mohammad Marwan, McNall Rebecca J, Adang Michael J, Dean Donald H
The Protein Research Group, The Ohio State University, Columbus, Ohio, USA.
Appl Environ Microbiol. 2003 Sep;69(9):5343-53. doi: 10.1128/AEM.69.9.5343-5353.2003.
Bacillus thuringiensis mosquitocidal toxin Cry4Ba has no significant natural activity against Culex quinquefasciatus or Culex pipiens (50% lethal concentrations [LC(50)], >80,000 and >20,000 ng/ml, respectively). We introduced amino acid substitutions in three putative loops of domain II of Cry4Ba. The mutant proteins were tested on four different species of mosquitoes, Aedes aegypti, Anopheles quadrimaculatus, C. quinquefasciatus, and C. pipiens. Putative loop 1 and 2 exchanges eliminated activity towards A. aegypti and A. quadrimaculatus. Mutations in a putative loop 3 resulted in a final increase in toxicity of >700-fold and >285-fold against C. quinquefasciatus (LC(50) congruent with 114 ng/ml) and C. pipiens (LC(50) 37 ng/ml), respectively. The enhanced protein (mutein) has very little negative effect on the activity against Anopheles or AEDES: These results suggest that the introduction of short variable sequences of the loop regions from one toxin into another might provide a general rational design approach to enhancing B. thuringiensis Cry toxins.
苏云金芽孢杆菌杀蚊毒素Cry4Ba对致倦库蚊或尖音库蚊没有显著的天然活性（50%致死浓度[LC(50)]分别>80,000和>20,000 ng/ml）。我们在Cry4Ba结构域II的三个推定环中引入了氨基酸替换。对突变蛋白在四种不同的蚊虫种类上进行了测试，即埃及伊蚊、四斑按蚊、致倦库蚊和尖音库蚊。推定环1和2的交换消除了对埃及伊蚊和四斑按蚊的活性。推定环3中的突变分别使对致倦库蚊（LC(50)约为114 ng/ml）和尖音库蚊（LC(50) 37 ng/ml）的毒性最终增加了>700倍和>285倍。增强后的蛋白（突变蛋白）对按蚊或伊蚊的活性几乎没有负面影响：这些结果表明，将一种毒素环区域的短可变序列引入另一种毒素中，可能为增强苏云金芽孢杆菌Cry毒素提供一种通用的合理设计方法。
