Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka, 819-0395, Japan.
Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812-8581, Japan.
Biotechnol J. 2017 Dec;12(12). doi: 10.1002/biot.201700195. Epub 2017 Oct 20.
Conjugating enzymes into a large protein assembly often results in an enhancement of overall catalytic activity, especially when different types of enzymes that work cooperatively are assembled together. However, exploring the proper method to achieve protein assemblies with high stability and also to avoid loss of the function of each component for efficient enzyme clustering is remained challenging. Assembling proteins onto synthetic scaffolds through varied post-translational modification methods is particularly favored since the proteins can be site-specifically conjugated together with less activity loss. Here, a SpyCatcher polymer is prepared through catalytic reaction of horseradish peroxidase (HRP) and serves as a polymeric proteinaceous scaffold for construction of protein assemblies. Taking advantage of the favorable SpyCatcher-SpyTag interaction, SpyTagged proteins can be easily assembled onto the polymeric SpyCatcher scaffold with controllable binding ratio and site specificity. Firstly, the feasibility of construction of ratio-controllable binary artificial hemicellulosomes by assembling endoxylanase and arabinofuranosidase is explored. This construct achieves higher sugar conversion than that of the free enzymes when the proportion of arabinofuranosidase is high, because the close spatial proximity of the enzymes allows them to work in a synergistic manner. Another application for biosensing is developed by conjugating SpyTagged Nanoluc and protein G onto SpyCatcher polymer. Due to the protein clustering effect, an amplified luminescent intensity is achieved by the resulting conjugates than chimera protein of Nanoluc and protein G in ovalbumin detection in ELISA.
将缀合酶组装到一个大型蛋白质组装体中通常会增强整体催化活性，特别是当协同作用的不同类型的酶组装在一起时。然而，探索实现具有高稳定性的蛋白质组装体的适当方法，同时避免每个组件的功能丧失以实现有效的酶聚类仍然具有挑战性。通过各种翻译后修饰方法将蛋白质组装到合成支架上特别受欢迎，因为可以特异性地将蛋白质连接在一起，而不会导致活性损失。在这里，通过辣根过氧化物酶（HRP）的催化反应制备了 SpyCatcher 聚合物，并用作构建蛋白质组装体的聚合蛋白质支架。利用有利的 SpyCatcher-SpyTag 相互作用，可以将 SpyTagged 蛋白质轻松组装到聚合物 SpyCatcher 支架上，具有可控的结合比和位点特异性。首先，通过组装内切木聚糖酶和阿拉伯呋喃糖苷酶探索构建比可控的二元人工半纤维素酶的可行性。当阿拉伯呋喃糖苷酶的比例较高时，该构建体实现了比游离酶更高的糖转化率，因为酶的紧密空间接近允许它们以协同方式工作。通过将 SpyTagged Nanoluc 和蛋白 G 缀合到 SpyCatcher 聚合物上，开发了另一种用于生物传感的应用。由于蛋白质聚类效应，与在卵清白蛋白检测中的 Nanoluc 和蛋白 G 的嵌合蛋白相比，所得缀合物实现了放大的发光强度。
