ACP Synthase (4´-phosphopantetheinyl transferase) catalyzes the covalent transfer of substituents from derivatized coenzyme A (CoA) to ACP-tagged fusion proteins exposed on the surface of living cells. The 25 nmoles of ACP Synthase provided is sufficient to make 25 ml of a 1 µM ACP-tag fusion protein labeling solution.
The ACP-tag and MCP-tag are small tags (8 kDa) based on the acyl carrier protein (ACP). MCP-tag contains two mutations (D36-T36 and D39-G39). Both allow the specific, covalent attachment of virtually any molecule to a protein of interest. Substrates for labeling are derivatives of coenzyme A (CoA). In the labeling reaction, the substituted phosphopantetheine group of CoA is covalently attached to a conserved serine residue on the ACP-tag or the MCP-tag by a phosphopantetheine transferase (SFP Synthase or ACP Synthase). Having no cysteines, the ACP-tag and the MCP-tag are particularly suited for specifically labeling cell-surface proteins, and should be useful for labeling secreted proteins with disulfide bridges such as antibodies.
While ACP Synthase will label predominantly the ACP-tag, SFP Synthase (NEB #P9302) will label both ACP-tag and MCP-tag. This principle can be employed for sequential dual labeling of two different proteins that localize to the cell surface. Cells co-expressing one ACP-tag fusion protein and one MCP-tag fusion protein can be incubated with ACP Synthase and one CoA substrate followed by labeling with SFP Synthase and a different CoA substrate. There are two steps to using this system: cloning and expression of the protein of interest as an ACP-tag fusion, and labeling of the fusion protein using the ACP Synthase with the CoA substrate of choice. In this document, the labeling of fusion proteins with CoA substrates is described. The cloning of ACP-tag protein fusions is described in the documentation supplied with the ACP-tag plasmids.
京公网安备11010802025653 版权所有：北京逸优科技有限公司
