Polymeric SpyCatcher Scaffold Enables Bioconjugation in a Ratio-Controllable Manner
Polymeric SpyCatcher Scaffold Enables Bioconjugation in a Ratio-Controllable Manner
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DOI：
10.1002/biot.201700195
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发表时间：
2017-12-01
影响因子：
4.7
通讯作者：
Kamiya, Noriho
中科院分区：
文献类型：
--
作者：
Jia, Lili;Minamihata, Kosuke;Kamiya, Noriho
将酶结合到一个大的蛋白质组合中通常会导致整体催化活性的提高，特别是当协同工作的不同类型的酶组合在一起时。然而，探索合适的方法来获得高稳定性的蛋白质组装，同时避免每个组分的功能损失以实现高效的酶聚类仍然是具有挑战性的。通过不同的翻译后修饰方法将蛋白质组装到合成支架上是特别受欢迎的，因为蛋白质可以在活性损失较小的情况下定点连接在一起。在这里，Spycatcher聚合物是通过催化辣根过氧化物酶(HRP)的反应而制备的，并作为构建蛋白质组件的聚合物蛋白质支架。利用SpyCatcher-SpyTag良好的相互作用，SpyTagge蛋白可以很容易地组装到聚合性SpyCatcher支架上，结合比例和位点特异性可控。首先，探讨了利用内切木聚糖酶和阿拉伯呋喃糖苷酶构建比例可控的二元人工半纤维素体的可行性。当阿拉伯呋喃糖苷酶的比例较高时，这种结构实现了比游离酶更高的糖转化率，因为酶的空间接近使它们能够以协同方式工作。另一种生物传感应用是通过将SpyTagated Nanolc和蛋白G偶联到SpyCatcher聚合物上。由于蛋白质的聚集效应，所得偶联物在检测卵清蛋白时比纳米蛋白和G蛋白的嵌合体蛋白具有更大的发光强度。
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.