Topology Engineering of Proteins in Vivo Using Genetically Encoded, Mechanically Interlocking SpyX Modules for Enhanced Stability.
Topology Engineering of Proteins in Vivo Using Genetically Encoded, Mechanically Interlocking SpyX Modules for Enhanced Stability.
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使用基因编码的机械联锁 SpyX 模块对体内蛋白质进行拓扑工程以增强稳定性
DOI：
10.1021/acscentsci.7b00104
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发表时间：
2017-05-24
影响因子：
18.2
通讯作者：
Zhang WB
中科院分区：
文献类型：
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作者：
Liu D;Wu WH;Liu YJ;Wu XL;Cao Y;Song B;Li X;Zhang WB
重组蛋白传统上限于线性构型。在这里，我们报告在体内蛋白质拓扑结构工程使用高效，机械联锁SpyX模块命名为AXB和BXA。SpyX模块是由p53 dim（X）、SpyTag（A）和SpyCatcher（B）组成的蛋白质结构域。p53 dim引导两条新生蛋白质链的缠绕，然后在SpyTag和SpyCatcher之间形成自催化异肽键以实现互锁，从而产生各种骨架拓扑结构。AXB或BXA的直接表达产生具有不同环大小的蛋白质链烷。如果目的蛋白融合到SpyX模块的N-或C-末端，则获得含有SpyX模块的重组蛋白作为机械互锁的专性二聚体，或者如果蛋白融合到N-和C-末端，则获得含有SpyX模块的重组蛋白作为星星蛋白。作为实例，证明了（GB 1）2（其中GB 1代表链球菌蛋白G的免疫球蛋白结合结构域B1）和四臂弹性蛋白样星星蛋白的二聚体的细胞合成。不同构建体中的连接效率的比较揭示了BXA通常比AXB有效得多，AXB通过空间中三个结构域的排列而合理化。机械互锁引起相当大的稳定性增强。AXB和BXA的熔点都比线性对照高约20 °C，BXA链烷的熔点比环状对照BX 'A高约2 °C。值得注意的是，四臂弹性蛋白样星星蛋白表现出对胰蛋白酶消化的显著耐受性。SpyX模块通过“组装-反应”协同作用提供了一种方便和通用的方法来构建非常规蛋白质拓扑结构，这为通过拓扑工程增强稳定性和功能强化开辟了蛋白质科学的新视野。
Recombinant proteins are traditionally limited to linear configuration. Herein, we report in vivo protein topology engineering using highly efficient, mechanically interlocking SpyX modules named AXB and BXA. SpyX modules are protein domains composed of p53dim (X), SpyTag (A), and SpyCatcher (B). The p53dim guides the intertwining of the two nascent protein chains followed by autocatalytic isopeptide bond formation between SpyTag and SpyCatcher to fulfill the interlocking, leading to a variety of backbone topologies. Direct expression of AXB or BXA produces protein catenanes with distinct ring sizes. Recombinant proteins containing SpyX modules are obtained either as mechanically interlocked obligate dimers if the protein of interest is fused to the N- or C-terminus of SpyX modules, or as star proteins if the protein is fused to both N- and C-termini. As examples, cellular syntheses of dimers of (GB1)2 (where GB1 stands for immunoglobulin-binding domain B1 of streptococcal protein G) and of four-arm elastin-like star proteins were demonstrated. Comparison of the catenation efficiencies in different constructs reveals that BXA is generally much more effective than AXB, which is rationalized by the arrangement of three domains in space. Mechanical interlocking induces considerable stability enhancement. Both AXB and BXA have a melting point ∼20 °C higher than the linear controls and the BXA catenane has a melting point ~2 °C higher than the cyclic control BX’A. Notably, four-arm elastin-like star proteins demonstrate remarkable tolerance against trypsin digestion. The SpyX modules provide a convenient and versatile approach to construct unconventional protein topologies via the “assembly-reaction” synergy, which opens a new horizon in protein science for stability enhancement and function reinforcement via topology engineering.