Chen Huayou, Wu Bangguo, Zhang Tianxi, Jia Jinru, Lu Jian, Chen Zhi, Ni Zhong, Tan Tianwei
Institute of life sciences, Jiangsu University, Xuefu Road No.301, Zhenjiang, 212013, People's Republic of China.
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
Appl Biochem Biotechnol. 2017 May;182(1):168-180. doi: 10.1007/s12010-016-2318-y. Epub 2016 Dec 8.
In fusion protein design strategies, the flexibility and length of linkers are important parameters affecting the bioactivity of multifunctional proteins. A series of fusion proteins with different linkers were constructed. The effect of temperature, pH, and organic solvents was investigated on the enzymatic activity. Fusion proteins with P1(PTPTPT) and P2((PTPTPT)) linkers remained highly active with wide temperature range. At pH 9.6, the relative activity of fusion proteins with (PTPTPT) and S2(EGKSSGSGSESKST) linkers was 70 and 62 % (1.75 and 1.5 times of that of non-linker ones). Fusion proteins with S3((GGGGS)) linker retained 55 % activity after 5 h of incubation at 80 °C (1.2-fold of that of non-linker fusion proteins and 1.9-fold of GGGGS-linker fusion proteins). Finally, the relative activity of fusion proteins having different linkers was increased with 20 % dimethyl sulfoxide (DMSO) and methanol; relative activity of fusion proteins with EGKSSGSGSESKST linkers was enhanced 1.5- and 2.2-fold, respectively. These results suggest that longer flexible linker can enhance the activity and stability of displayed esterase than shorter flexible linker. Optimizing peptide linkers with length, flexibility, and amino acid composition could improve the thermostability and activity of the displayed enzyme.
在融合蛋白设计策略中，连接子的灵活性和长度是影响多功能蛋白生物活性的重要参数。构建了一系列带有不同连接子的融合蛋白。研究了温度、pH值和有机溶剂对酶活性的影响。带有P1（PTPTPT）和P2（(PTPTPT)）连接子的融合蛋白在较宽温度范围内保持高活性。在pH 9.6时，带有（PTPTPT）和S2（EGKSSGSGSESKST）连接子的融合蛋白的相对活性分别为70%和62%（是非连接子融合蛋白的1.75倍和1.5倍）。带有S3（(GGGGS)）连接子的融合蛋白在80℃孵育5小时后保留55%的活性（是非连接子融合蛋白的1.2倍，是GGGGS连接子融合蛋白的1.9倍）。最后，不同连接子的融合蛋白在20%二甲基亚砜（DMSO）和甲醇存在下相对活性增加；带有EGKSSGSGSESKST连接子的融合蛋白相对活性分别提高了1.5倍和2.2倍。这些结果表明，较长的柔性连接子比较短的柔性连接子能增强展示酯酶的活性和稳定性。优化连接子的长度、灵活性和氨基酸组成可以提高展示酶的热稳定性和活性。
