Zheng Yanjun, Chen Fei, Frank Saskia, Quispe Haro Juan José, Wegner Seraphine V
Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, 48149, Münster, Germany.
Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, 410083, China.
Small. 2024 Dec;20(52):e2405687. doi: 10.1002/smll.202405687. Epub 2024 Oct 18.
Protein photolithography is an invaluable tool for generating protein microchips and regulating interactions between cells and materials. However, the absence of light-responsive molecules that allow for the copatterning of multiple functional proteins with biocompatible visible light poses a significant challenge. Here, a new approach for photopatterning three distinct proteins on a single surface by using green, red, and far-red light is reported. The cofactor of the green light-sensitive protein CarH is engineered such that it also becomes sensitive to red and far-red light. These new cofactors are shown to be compatible with two CarH-based optogenetic tools to regulate bacterial cell-cell adhesions and gene expression in mammalian cells with red and far-red light. Further, by incorporating different CarH variants with varying light sensitivities in layer-by-layer (LbL) multiprotein films, specific layers within the films, along with other protein layers on top are precisely removed by using different colors of light, all with high spatiotemporal accuracy. Notably, with these three distinct colors of visible light, it is possible to incorporate diverse proteins under mild conditions in LbL films based on the reliable interaction between Ni- nitrilotriacetic acid (NTA) groups and polyhistidine-tags (His-tags)on the proteins and their subsequent photopatterning. This approach has potential applications spanning biofabrication, material engineering, and biotechnology.
蛋白质光刻技术是用于制造蛋白质微芯片以及调节细胞与材料之间相互作用的一项宝贵工具。然而，缺乏能够实现多种功能蛋白质与生物相容性可见光共图案化的光响应分子构成了一项重大挑战。在此，报道了一种通过使用绿光、红光和远红光在单个表面上对三种不同蛋白质进行光图案化的新方法。对绿光敏感蛋白CarH的辅因子进行了工程改造，使其对红光和远红光也变得敏感。这些新的辅因子被证明与两种基于CarH的光遗传学工具兼容，可分别用红光和远红光调节细菌细胞间粘附以及哺乳动物细胞中的基因表达。此外，通过在逐层（LbL）多蛋白膜中纳入具有不同光敏感性的不同CarH变体，利用不同颜色的光以高时空精度精确去除膜内特定层以及顶部的其他蛋白层。值得注意的是，利用这三种不同颜色的可见光，基于蛋白质上的镍 - 次氮基三乙酸（NTA）基团与多组氨酸标签（His - tags）之间可靠的相互作用及其后续的光图案化，有可能在温和条件下将多种蛋白质纳入LbL膜中。这种方法在生物制造、材料工程和生物技术等领域具有潜在应用。
