Linkers are critical components of fusion proteins, as they physically separate individual domains to enable each to fold and retain function. The role of peptide linker properties was investigated for fusions of a leucine zipper immobilization domain (Z_E) to a chimeric amine dehydrogenase (AmDH) or a formate dehydrogenase (cbFDH). A linker library was developed, which varied in length, orientation, and proline content, as a way to vary stiffness. Fusion proteins were characterized by melting temperature, immobilization ability, cofactor binding, and kinetic activity. The best linker candidate for each enzyme was tested in a dual‐functionality assay, where enzymatic activity of fusions immobilized in protein‐inorganic supraparticles was greater than 80% after washing. The best linker for AmDH was completely different than that for cbFDH. This work highlights the need to experimentally assess linker properties in the design of new fusion proteins and provides a linker library for this purpose. © 2018 American Institute of Chemical Engineers AIChE J, 64: 2934–2946, 2018

中文翻译：

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肽接头长度和组成对亮氨酸拉链-酶融合蛋白固定化和催化的影响

接头是融合蛋白的关键组成部分，因为它们物理上将各个域分开，从而使每个域都能折叠并保持功能。研究了肽接头性质对亮氨酸拉链固定结构域（Z _E）融合的作用）转化为嵌合胺脱氢酶（AmDH）或甲酸酯脱氢酶（cbFDH）。开发了一个连接子库，它的长度，方向和脯氨酸含量各不相同，以此来改变刚度。融合蛋白的特征在于解链温度，固定能力，辅因子结合和动力学活性。在双重功能测定中测试了每种酶的最佳候选连接物，其中固定在蛋白质-无机超颗粒中的融合物在洗涤后的酶活性大于80％。AmDH的最佳接头与cbFDH的接头完全不同。这项工作强调了在新融合蛋白设计中需要实验评估接头性质的需要，并为此目的提供了一个接头库。©2018美国化学工程师学会AIChE J，64：2934–2946，2018