Innovative synthesis and purification method of fluorescent and bifunctional substrates for self-labelling protein tags

Self-labelling protein tags, including SNAP-tag, HaloTag, and CLIP-tag, have revolutionized molecular biology by enabling precise visualization and manipulation of proteins within living cells. These systems use suicidal proteins that react with synthetic substrates to facilitate the attachment of fluorescent dyes, which offer advantages such as flexibility in dye selection and compatibility with advanced imaging techniques like fluorescence microscopy and fluorimetry. This paper presents a new methodology for the synthesis and high-yield purification of fluorescent probes for SNAP-tag, CLIP-tag, and HaloTag, while also investigating their kinetic parameters. A key innovation is the design of a bifunctional molecule capable of selectively cross-linking SNAP-tag and HaloTag proteins, overcoming potential cross-reactivity between SNAP-tag and CLIP-tag. The synthesis was achieved with improved reaction yields using flash chromatography, replacing traditional HPLC methods, and characterized using NMR spectroscopy and mass spectrometry. Biochemical assays demonstrated the suitability of the new substrates for various AGTs and HaloTag, with kinetic studies revealing their efficacy at different temperatures. Additionally, the bifunctional linker was shown to enable the simultaneous capture of SNAP-tag and HaloTag proteins, with fluorescence assays and native gel electrophoresis confirming the formation of protein complexes. These advancements in substrate production, characterization, and cross-linking design enhance the versatility of SLPs, offering new opportunities for studying protein dynamics and interactions in live cells. This work paves the way for further biotechnological applications of these powerful tools.