Department of Biomolecular Innovation, Institute for Biomedical Sciences, Shinshu University, Ueda, Nagano, Japan; Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano, Japan.
Methods Enzymol. 2021;647:209-230. doi: 10.1016/bs.mie.2020.10.003. Epub 2020 Nov 18.
The construction of recombinant fusion/chimeric proteins has been widely used for expression of soluble proteins and protein purification in a variety of fields of protein engineering and biotechnology. Fusion proteins are constructed by the linking of two protein domains with a peptide linker. The selection of a linker sequence is important for the construction of stable and bioactive fusion proteins. Empirically designed linkers are generally classified into two categories according to their structural features: flexible linkers and rigid linkers. Rigid linkers with the α-helix-forming sequences A(EAAAK)A (n=2-5) were first designed about two decades ago to control the distance between two protein domains and to reduce their interference. Thereafter, the helical linkers have been applied to the construction of many fusion proteins to improve expression and bioactivity. In addition, the design of fusion proteins that self-assemble into supramolecular complexes is useful for nanobiotechnology and synthetic biology. A protein that forms a self-assembling oligomer was fused by a rigid helical linker to another protein that forms another self-assembling oligomer, and the fusion protein symmetrically self-assembled into a designed protein nanoparticle or nanomaterial. Moreover, to construct chain-like polymeric nanostructures, extender protein nanobuilding blocks were designed by tandemly fusing two dimeric de novo proteins with helical or flexible linkers. The linker design of fusion proteins can affect conformation and dynamics of self-assembling nanostructures. The present review and methods focus on useful helical linkers to construct bioactive fusion proteins and protein-based nanostructures.
重组融合/嵌合蛋白的构建已广泛应用于蛋白质工程和生物技术的多个领域中可溶性蛋白的表达和蛋白纯化。融合蛋白通过肽接头将两个蛋白结构域连接起来构建而成。接头序列的选择对于构建稳定且具有生物活性的融合蛋白非常重要。经验设计的接头通常根据其结构特征分为两类：柔性接头和刚性接头。刚性接头具有α-螺旋形成序列 A(EAAAK)A（n=2-5），大约二十年前首次被设计用于控制两个蛋白结构域之间的距离并减少它们之间的干扰。此后，螺旋接头已被应用于许多融合蛋白的构建中，以提高表达和生物活性。此外，设计自组装成超分子复合物的融合蛋白对于纳米生物技术和合成生物学非常有用。通过刚性螺旋接头将形成自组装寡聚物的蛋白与另一个形成另一种自组装寡聚物的蛋白融合，融合蛋白对称地自组装成设计的蛋白纳米颗粒或纳米材料。此外，为了构建链状聚合体纳米结构，通过串联融合两个具有螺旋或柔性接头的二聚体从头蛋白来设计扩展蛋白纳米构建块。融合蛋白的接头设计可以影响自组装纳米结构的构象和动力学。本综述和方法重点介绍了用于构建具有生物活性的融合蛋白和基于蛋白的纳米结构的有用的螺旋接头。
