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Sep 17, 2012

Technology Enables Reversible, Repeatable Protein Labeling

  • A method that allows proteins to be tagged reversibly and repeatedly with a wide range of probes could revolutionize the way proteins function, biosynthesis and interactions are studied, researchers claim. The method, developed by a team at the University of California, San Diego, is based on Acyl carrier protein (ACP) labeling with 4’-phosphopantetheine (PPant) conjugated to a desired tag using its transferase (PPTase). This technique has been successfully used for protein isolation, and for protein visualization, and functional and structural studies, explain Michael D. Burkart, M.D., and colleagues. However, it’s not possible to easily reverse PPant attachment.

    To overcome this problem, the researchers used ACP hydrolase (AcpH), a phosphodiesterase from Pseudomonas aeruginosa 13, and Sfp, a PPTase from Bacillus subtilis, to swap different PPant-conjugated small molecules on free ACP and ACP fusion proteins. Their method, the culmination of some 10 years of research, was devised as part of their study of fatty acid metabolism and the biosynthesis of other natural products, in particular the production of biofuels from engineered algae. Importantly, “Without this tool, we would really have very limited ways of studying the dynamics of these fundamental metabolic processes,” professor Burkart states. “This opened the door for us to finally examine in detail the fatty acid biosynthesis shared by algae, which you have to understand if you want to engineer ways to improve the quantity of oil that’s made by algae. Importantly, NMR analysis confirmed theat the process of chemically removing and attaching probes doesn’t damage the protein. “We’ve shown that we can do this iteratively, at least four or five times, without any degradation of the protein,” professor Burkart claims. “The protein remains very stable and can be studied very easily.”

    The investigators hope the ability to attach and remove probes to proteins will facilitate the study of a range of biosynthetic processes and pathways. “Given the multitude of existing opportunities for ACP labeling, particularly in work involving fusion-protein applications and natural-product biosynthetic studies, we believe that providing a reversible methodology will provide markedly improved flexibility for rapid modification of protein species,” the UCSD researchers write in their published paper in Nature Methods. “There’s a great interest now in synthetic biology, using these pathways to make new antibiotics or anti-cancer drugs,” professor Burkart adds. “They’re all regulated by these same types of interactions."

    The method should also have wide-ranging applications as a laboratory tool to visualize and track proteins on cells, and manipulate them in the extracellular environment, the team believes. “One could attach a tag, such as biotin, that would allow the protein to be purified. Then one can clip off the tag and attach a fluorescent molecule to monitor protein interaction with other molecular partners,” professor Burkart suggests. “The method could also be used for studying living cells, such as observing protein expression levels throughout the cellular life cycle.”

    The authors report their technology in a paper titled “Reversible labeling of native and fusion-protein motifs.”


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