Three‐Color Protein Photolithography with Green, Red, and Far‐Red Light

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 Ni²⁺- 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.