Description: The ACP-tag is a novel tool for the specific, covalent attachment of virtually any molecule to a cell surface protein of interest, providing simplicity and extraordinary versatility to the imaging of proteins in live and fixed cells and to the study of proteins in vitro. The creation of a single gene construct yields a tagged fusion protein capable of covalent derivatization with a variety of functional groups, including fluorophores and biotin. This system provides a powerful and unique tool to study the role of cell surface proteins in a variety of highly dynamic processes, including receptor internalization, turnover and complex formation.
The ACP-tag and the related MCP-tag are small protein tags (77 amino acids, 8 kDa) based on the acyl carrier protein from E. coli. Both can be enzymatically modified with fluorophores, biotin etc. using substrates that are derivatives of coenzyme A (CoA). In the labeling reaction, the substituted phosphopantetheine group of the CoA substrate is covalently attached to a conserved serine residue of the ACP-tag or the MCP-tag by a 4´-phosphopantetheinyl transferase (SFP Synthase from B. subtilis, or ACP Synthase from E. coli) (Figure 1). The ACP-tag has a number of features that make it ideal for a variety of protein labeling applications. The rate of the reaction of the synthase with CoA derivatives is largely independent of the nature of the synthetic probe attached to CoA, permitting the labeling of ACP and MCP fusion proteins with a wide variety of functional groups (Figure 2). The ability to turn on the signal at will allows time-resolved analysis of protein trafficking and receptor internalization. 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. Finally, the availability of orthogonal protein labeling systems from NEB permits simultaneous labeling of multiple proteins in a single cell (SNAP-tag, a variant of the human DNA repair enzyme hAGT that transfers a label onto itself from O6-benzylguanine substrates, and CLIP-tag, a SNAP-tag variant that transfers a label onto itself from O2-benzylcytosine substrates).
While the ACP Synthase (NEB #P9301) will modify predominantly the ACP-tag, the included SFP Synthase will efficiently label both the ACP-tag and MCP-tag (the MCP-tag contains two mutations relative to the ACP-tag, D36T and D39G, which abolish recognition by the ACP Synthase while preserving recognition by SFP Synthase). 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 are first incubated with ACP Synthase and one CoA substrate to label the ACP-tag, followed by incubation with the SFP Synthase and a different CoA substrate to label the MCP-tag.
The ACP-Surface Starter Kit contains a mammalian expression plasmid (pACP-tag(m)-2) encoding the ACP-tag flanked by restriction sites for cloning a gene of interest, SFP Synthase, 1 M MgCl2 and two non-cell-permeable fluorescent CoA substrates. A positive control plasmid (pACP-ADRβ2), encoding an ACP-tagged protein (Beta-2 Adrenergic Receptor) with a well-characterized cell surface localization, is also included. There are two steps to using this system: subcloning and expression of the protein of interest as an ACP-tag fusion, and enzymatically labeling the fusion with the CoA substrate of choice.
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