Engineering a Blue Light Inducible SpyTag System (BLISS)
Engineering a Blue Light Inducible SpyTag System (BLISS)
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DOI：
10.1021/jacs.1c03198
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发表时间：
2021-06-02
影响因子：
15
通讯作者：
Chen, Wilfred
中科院分区：
文献类型：
--
作者：
Hartzell, Emily J.;Terr, Justin;Chen, Wilfred
SpyCatcher/SpyTag蛋白偶联系统最近因其快速的动力学和在体外和细胞内环境下生物有利的条件下的高产量而受到欢迎。通过引入在空间和时间上控制共轭事件的能力，可以扩展该系统的用途。受自然界中光感受器蛋白的启发，我们设计了一种将光依赖性整合到蛋白质偶联反应中的方法。燕麦的光-氧-电压结构域2(AsLOV2)在其c-末端Jα-螺旋结构中发生了戏剧性的构象变化，以响应蓝光。通过将SpyTag插入到Jα-螺旋的不同位置，我们创建了一个蓝光诱导SpyTag系统(BLISS)。在这种设计中，SpyTag被阻止在黑暗中与SpyCatcher反应，但在蓝光照射下，AsLOV2的Jα螺旋脱离对接，暴露出SpyTag。我们测试了几个插入位点，并对动力学进行了表征。我们发现了三个动态范围超过15的变异体，它们在不同的浓度范围内都有活性。可以使用具有不同反应动力学的SpyCatcher变体来调整这些参数。此外，通过移除光线，该反应可以瞬间熄灭。我们通过两种荧光蛋白的光致变色演示了这种光控制机制的空间方面。该系统为许多其他生物制造和光遗传学应用提供了机会。
The SpyCatcher/SpyTag protein conjugation system has recently exploded in popularity due to its fast kinetics and high yield under biologically favorable conditions in both in vitro and intracellular settings. The utility of this system could be expanded by introducing the ability to spatially and temporally control the conjugation event. Taking inspiration from photoreceptor proteins in nature, we designed a method to integrate light dependency into the protein conjugation reaction. The light-oxygen-voltage domain 2 of Avena sativa (AsLOV2) undergoes a dramatic conformational change in its c-terminal J alpha-helix in response to blue light. By inserting SpyTag into the different locations of the J alpha-helix, we created a blue light inducible SpyTag system (BLISS). In this design, the SpyTag is blocked from reacting with the SpyCatcher in the dark, but upon irradiation with blue light, the J alpha-helix of the AsLOV2 undocks to expose the SpyTag. We tested several insertion sites and characterized the kinetics. We found three variants with dynamic ranges over 15, which were active within different concentration ranges. These could be tuned using SpyCatcher variants with different reaction kinetics. Further, the reaction could be instantaneously quenched by removing light. We demonstrated the spatial aspect of this light control mechanism through photopatterning of two fluorescent proteins. This system offers opportunities for many other biofabrication and optogenetics applications.