Georgyi V. Los1, Natasha Karassina1, Chad Zimprich1, Mark G. McDougal2, Randall Learish1, Soshana Svendsen1, Dieter H. Klaubert2, Keith Wood1, Robert F. Bulleit1
1Promega Corporation, 2800 Woods Hollow Rd. Madison, WI 53711
2Promega Biosciences Inc. 277 Granada Dr., San Luis Obispo, CA 93401
Development of novel reporter systems for live cell imaging is essential for further advancement of life science research and drug discovery. Recently we have described a novel technology for site-specific covalent tethering of synthetic ligands to a novel reporting protein, HaloTag® Protein, in vitro and in living cells. The reporter protein is a genetically engineered catalytically inactive derivative of hydrolase. The ligands are small chemical tags capable of carrying a variety of functionalities, such as fluorescent labels, affinity handles, or attachments to a solid phase. The covalent bond forms rapidly under general physiological conditions, is highly specific, and essentially irreversible. Here we demonstrate that HaloTag® System is well tolerated by mammalian cells. Fusion of different proteins to HaloTag® Protein does not interfere with protein function. The open architecture of the technology ensures interchangeability of ligands, thereby facilitating a variety of functional studies (including imaging at different wavelengths and temporal or spatial separation of protein pools) without requiring changes to the underlying genetic construct. The stability of the bond between HaloTag® Protein and HaloTag® Ligand allows imaging of live cells during long periods of time, imaging of fixed cells, and multiplexing with different cell/protein analysis techniques. We also demonstrate that HaloTag® technology can be used to study dynamic cellular events in 96- and 384-well formats. The technology complements existing methods and provides new options for cellular analysis and potentially High Content Screening.