Department of Bioengineering, University of California San Diego, La Jolla, CA, United States; Department of Chemical Engineering, Institute of Biochemical Engineering, Beijing, China; Key Laboratory for Industrial Biocatalysis, Ministry of Education, Beijing, China.
Department of Chemical Engineering, Institute of Biochemical Engineering, Beijing, China; Key Laboratory for Industrial Biocatalysis, Ministry of Education, Beijing, China; Center for Synthetic and Systems Biology, Tsinghua University, Beijing, China.
Methods Enzymol. 2021;647:23-49. doi: 10.1016/bs.mie.2020.12.004. Epub 2020 Dec 26.
Linkers play essential roles in the engineering of fusion proteins, and have been extensively demonstrated to affect protein properties such as expression level, solubility, and biological functions. For linker design and optimization, one of the key factors is the flexibility or rigidity of linkers, which describes the tendency of a linker to maintain a stable conformation when expressed, and can directly contribute to the physical distance between domains of a fusion protein. In this chapter, we discuss the design and engineering of linkers in fusion proteins, and describe a library-based method for optimization of linker flexibility. This approach is based on chimeric linkers, which are composed of both flexible and rigid (helix-forming) linker motifs. We demonstrate that the chimeric linker library capable of controlling the flexibility in a wide range can fill the gap between flexible and rigid linkers by molecular dynamics simulation and fluorescence resonance energy transfer experiments, as well as its applications in fusion protein optimization.
接头在融合蛋白的工程化中起着至关重要的作用，并且已广泛证明会影响蛋白质的性质，如表达水平、溶解度和生物功能。对于接头设计和优化，关键因素之一是接头的柔韧性或刚性，这描述了接头在表达时保持稳定构象的趋势，并且可以直接影响融合蛋白结构域之间的物理距离。在本章中，我们讨论了融合蛋白中接头的设计和工程，并描述了一种基于文库的优化接头柔韧性的方法。该方法基于嵌合接头，它由柔性和刚性（形成螺旋）接头基序组成。我们通过分子动力学模拟和荧光共振能量转移实验证明，能够在广泛范围内控制柔韧性的嵌合接头文库可以填补柔性和刚性接头之间的空白，并展示了其在融合蛋白优化中的应用。
