Description:
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 O
6-benzylguanine-containing and O
2-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 O
6-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.