Recently-developed protein tags enable the specific covalent attachment of synthetic ligands, incorporating fluorophores or other substituted groups, to fusion proteins containing these tags. For example, SNAP and CLIP tags bind O6-benzylguanine-containing and O2-benzylcytosine containing ligands respectively, which can be derivatized with a wide variety of labels, including fluorescent dyes, affinity probes, and cross-linkers. This system provides a powerful tool to study a variety of highly dynamic processes within cells, including protein trafficking, turnover, and complex formation. However, a substantial limitation to this approach is that labeling is irreversible, due to the formation of a covalent bond between the probe and the protein tag.
The inventors have developed ligands that incorporate a disulfide linkage between the O6-benzylguanine moiety and the label, allowing selective release of the label from the tagged protein when treated with a reducing agent. The inventors have shown that use of these ligands in conjunction with cell-impermeable reducing agents allows visualization of internalization and trafficking in live cells; these ligands may also be used in other applications in which a cleavable label would be desirable, such as protein purification. This strategy is also applicable to other covalent protein tags, such as the ACP/MCP protein tag system.
- Visualization of dynamic processes within cells, including protein trafficking, turnover, and complex formation
- Live cell imaging
- Protein purification
- Allows for selective release of label
- Accommodates intra- or extra-cellular labeling, and dual labeling
- Ligands may be derivatized with a wide variety of labels, including fluorescent dyes, affinity probes, and cross-linkers
- Lower background fluorescence and higher contrast than other systems, such as FlAsH