Background

Naturally photoswitchable proteins act as a powerful tool for the spatial and temporal control of biological processes by inducing the formation of a photodimerizer. In this study, a method for the precise and reversible inducible self-assembly of dodecamer nitrilase in vivo (in Escherichia coli) and in vitro (in a cell-free solution) was developed by means of the photoswitch-improved light-inducible dimer (iLID) system which could induce protein–protein dimerization.

Results

Nitrilase was fused with the photoswitch protein AsLOV2-SsrA to achieve the photocontrolled self-assembly of dodecamer nitrilase. The fusion protein self-assembled into a supramolecular assembly when illuminated at 470 nm. Scanning electron microscopy showed that the assembly formed a circular sheet structure. Self-assembly was also induced by light in E. coli. Dynamic light scattering and turbidity assay experiments showed that the assemblies formed within a few seconds under 470-nm light and completely disassembled within 5 min in the dark. Assembly and disassembly could be maintained for at least five cycles. Both in vitro and in vivo, the assemblies retained 90% of the initial activity of nitrilase and could be reused at least four times in vitro with 90% activity.

Conclusions

An efficient method was developed for the photocontrolled assembly and disassembly of dodecamer nitrilase and for scaffold-free reversible self-assembly of multiple oligomeric enzymes in vivo and in vitro, providing new ideas and methods for immobilization of enzyme without carrier.

中文翻译：

---

十二胺腈水解酶的光控可逆自组装。

背景

通过诱导光二聚体的形成，天然的可光转换蛋白可作为功能强大的工具来对生物过程进行时空控制。在这项研究中，通过光开关改良的光诱导二聚体（在体内（在大肠杆菌中）和体外（在无细胞溶液中），开发了一种精确且可逆的十二聚体腈水解酶诱导自组装方法。 iLID）系统可以诱导蛋白质-蛋白质二聚化。

结果

将腈水解酶与光开关蛋白AsLOV2-SsrA融合，以实现十二聚腈水解酶的光控自组装。融合蛋白在470 nm照射时自组装成超分子组装体。扫描电子显微镜显示该组件形成圆形片状结构。大肠杆菌中的光也诱导了自组装。动态光散射和浊度测定实验表明，组件在470 nm的光下于几秒钟内形成，并在黑暗中5分钟内完全分解。组装和拆卸可至少维持五个周期。在体外和体内，该组件都保留了腈水解酶初始活性的90％，并且可以以90％的活性在体外重复使用至少四次。

结论

开发了一种有效的方法，用于光控组装和拆卸十二聚氰胺腈水解酶，以及在体内和体外无支架地进行多种寡聚酶的可逆自组装，从而提供了无需载体即可固定化酶的新思路和方法。