This control plasmid contains a glycosylphosphatidylinositol (GPI) anchor sequence cloned upstream of the MCP-tag coding sequence in pMCP-tag(m). This sequence directs the fusion protein to the plasma membrane with the MCP-tag exposed on the outside surface, giving a localized control signal when labeled with CoA substrates. GPI is present in all eukaryotic cells where it anchors glycoproteins to the cell surface. The GPI anchor helps increase the mobility of membrane proteins thereby supporting signal transduction and cellular transport. In addition, the GPI anchor plays an important role in creating antigens on cell surfaces.
The MCP-tag is a small protein tag (8 kDa) based on the acyl carrier protein (ACP) containing two mutations (D36-T36 and D39-G39), for labeling cell membrane proteins. It allows the specific, covalent attachment of virtually any molecule to a protein of interest. MCP-tag substrates are derivatives of coenzyme A (CoA). In the labeling reaction, the substituted phosphopantetheine group of CoA is covalently attached to a conserved serine residue of the MCP-tag by a phosphopantetheine transferase (SFP Synthase). Having no cysteines, the MCP-tag is particularly suited for specifically labeling cell surface proteins, and should be useful for labeling secreted proteins with disulfide bridges such as antibodies.
There are two steps to using this system: sub cloning and expression of the protein of interest as an MCP-tag fusion, and labeling of the fusion protein using SFP Synthase with the substrate of choice.The cloning and expression of MCP-tag protein fusions is described in documents provided with the pMCP-tag(m) cloning plasmid. The labeling of fusion proteins with CoA substrates is described in the documentation supplied with CoA substrates and SFP Synthase.
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