高等学校化学学报

高等学校化学学报

• 研究论文 • 上一篇    下一篇

W544F定点突变提高苏云金杆菌Cry1Ac蛋白的稳定性

王发祥1, 夏立秋1,2, 丁学知1, 赵新民1, 单世平1, 莫湘涛1, 张友明1, 喻子牛2   

    1. 湖南师范大学生命科学学院, 微生物分子生物学湖南省重点实验室, 长沙 410081;
    2. 华中农业大学生命科学与技术学院, 农业微生物国家重点实验室, 武汉 430070
  • 收稿日期:2008-02-28 修回日期:1900-01-01 出版日期:2008-10-10 发布日期:2008-10-10
  • 通讯作者: 夏立秋

Stability Improvement of the Cry1Ac Protein by Site-directed Mutagenesis of W544F

WANG Fa-Xiang1, XIA Li-Qiu1,2*, DING Xue-Zhi1, ZHAO Xin-Min1, SHAN Shi-Ping1, MO Xiang-Tao1, ZHANG You-Ming1, YU Zi-Niu2   

    1. Key Laboratory for Molecular Biology of Microorganism of Hunan Province, College of Life Science, Hunan Normal University, Changsha 410081, China;
    2. National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
  • Received:2008-02-28 Revised:1900-01-01 Online:2008-10-10 Published:2008-10-10
  • Contact: XIA Li-Qiu

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被引次数

9

摘要： W544是Cry1Ac蛋白上独特于其它Cry类蛋白的一个氨基酸, 它与F578和F604一起组成一个“螺旋桨状”的疏水簇, 通过疏水相互作用维持蛋白的三维结构稳定. 本研究通过定点突变将W544保守地替换为苯丙氨酸, SDS-PAGE分析结果表明其纯化的原毒素对紫外照射、胰蛋白酶处理和室温存贮的稳定性相对于野生Cry1Ac都有一定程度的提高; 经原子力显微镜观察, 发现W544F产生的晶体两个顶点间的垂直距离比野生型Cry1Ac约长0.6 μm, 且晶体表面不及野生型光滑; 此外, W544F与野生Cry1Ac的杀虫活性相似, 但经过紫外光照射9 h后, 其保留的杀虫活性比野生型高4倍以上. W544F突变较好地解决了Cry1Ac毒素蛋白田间应用不持久的问题, 具有重要的应用价值.

关键词: Cry1Ac蛋白, 定点突变, 色氨酸, 苯丙氨酸, 稳定性

Abstract: One major problem in the field applications of Bacillus thuringiensis Cry toxins is that their exposure to sunlight can bring about reduced bioactivity, mainly because of the most vulnerable indole ring of tryptophan(W) residue. W544 is the unique tryptophan in the domain Ⅲ of Cry1Ac toxin, which forms part of a buried hydrophobic cluster involving the conserved F578 and F604, and are organized in a “propeller-like” pattern, playing a clear structural role in protein stability. In this study, W544 was conservatively substituted with phenylalanine(F) to determine whether its stability was affected in the resulting mutant. Comparative analysis results by SDS-PAGE show that the protoxin of W544F was more stable than the wild-type Cry1Ac, when treated with ultraviolet irradiation, trypsin and preserved at room temperature. The distance between two vertex of crystals of W544F were 0.6 μm longer and unsmooth when compared with that of the wild-type Cry1Ac under an atomic force microscope. Addionally the mutation W544F had similar insecticidal activity to wild-type Cry1Ac, but when treated with ultraviolet irradiation for 9 h, it still maintained more than 4 times higher toxicity against Helicoverpa armigera than the wild-type Cry1Ac. In conclusion, the W544F mutation can enhance the stability of the Cry1Ac protein and may contribute to solving the major problem of the field applications of Cry1Ac toxin.

Key words:

Cry1Ac protein, Site-directed mutagenesis, Tryptophan, Phenylalanine, Stability

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