Zhang Haibo, Zhou Fengli, Jiang Xinglin, Cao Mingle, Wang Shilu, Zou Huibin, Cao Yujin, Xian Mo, Liu Huizhou
a CAS Key Laboratory of Biobased Materials , Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences , Qingdao , China.
b Department of Chemistry & Chemical Biology , University of Marburg , Marburg , Germany.
Prep Biochem Biotechnol. 2016 Aug 17;46(6):552-8. doi: 10.1080/10826068.2015.1084637.
Spider dragline silk is a remarkably strong fiber with impressive mechanical properties, which were thought to result from the specific structures of the underlying proteins and their molecular size. In this study, silk protein 11R26 from the dragline silk protein of Nephila clavipes was used to analyze the potential effects of the special amino acids on the function of 11R26. Three protein derivatives, ZF4, ZF5, and ZF6, were obtained by site-directed mutagenesis, based on the sequence of 11R26, and among these derivatives, serine was replaced with cysteine, isoleucine, and arginine, respectively. After these were expressed and purified, the mechanical performance of the fibers derived from the four proteins was tested. Both hardness and average elastic modulus of ZF4 fiber increased 2.2 times compared with those of 11R26. The number of disulfide bonds in ZF4 protein was 4.67 times that of 11R26, which implied that disulfide bonds outside the poly-Ala region affect the mechanical properties of spider silk more efficiently. The results indicated that the mechanical performances of spider silk proteins with small molecular size can be enhanced by modification of the amino acids residues. Our research not only has shown the feasibility of large-scale production of spider silk proteins but also provides valuable information for protein rational design.
蜘蛛拖牵丝是一种具有卓越机械性能的超强纤维，其机械性能被认为源于基础蛋白质的特定结构及其分子大小。在本研究中，来自棒络新妇蛛拖牵丝蛋白的丝蛋白11R26被用于分析特定氨基酸对11R26功能的潜在影响。基于11R26的序列，通过定点诱变获得了三种蛋白质衍生物ZF4、ZF5和ZF6，在这些衍生物中，丝氨酸分别被半胱氨酸、异亮氨酸和精氨酸取代。在它们被表达和纯化后，测试了源自这四种蛋白质的纤维的机械性能。与11R26相比，ZF4纤维的硬度和平均弹性模量均提高了2.2倍。ZF4蛋白中二硫键的数量是11R26的4.67倍，这表明聚丙氨酸区域外的二硫键能更有效地影响蜘蛛丝的机械性能。结果表明，通过修饰氨基酸残基可以提高小分子蜘蛛丝蛋白的机械性能。我们的研究不仅证明了大规模生产蜘蛛丝蛋白的可行性，还为蛋白质合理设计提供了有价值的信息。
