Marjeta Urh1, Dan Simpson1, Nidhi Nath1, Jacqui Sankbeil1, Georgyi V. Los1, Chad Zimprich1, Natasha Karassina1, Randy Learish1, Rachel Freidman-Ohana1, Lance P. Encell1, Monika Wood1, Kate Qin Zhao1, Doug Storts1, Bob Bulleit1, Keith V. Wood1, Ji Zhu2, Mark McDougall2, Poncho Meisenheimer2 and Dieter H. Klaubert2
1Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711
2Promega Biosciences, Inc., 277 Granada Drive, San Luis Obispo, CA 93401
It is becoming clear that surface-based proteomics and protein microarrays will play an important role in the future of proteomics. Successful implementation of surface-based proteomics requires methods that enable stable attachment of proteins while maintaining three dimensional structure and activity. Here we describe a method for specific, covalent and oriented immobilization of proteins onto surfaces. The strategy is based on a fusion protein tag that is a catalytically inactive hydrolase (HaloTag®) designed to form a covalent bond with specific a ligand at a rate comparable to the rate of biotin:streptavidin interactions. We chemically modified different surfaces with the HaloTag® ligand, which allows immobilization of fusion proteins to those surfaces. The rapid and highly specific interaction between the protein and ligand allows immobilization of fusion proteins without the need for prior purification. To demonstrate that protein fusions immobilized via HaloTag® maintain their function, we analyzed the activities of several different fusion proteins. Using known protein:protein interactions, we show that immobilized proteins interact with their partners with expected specificity. Furthermore the immobilized proteins maintain greater enzymatic activity compared to randomly immobilized proteins. Thus the HaloTag® technology is well suited for in vitro analysis of protein activity. To further our understanding of intracellular processes such as signal transduction pathways, analysis of protein function in living cells is also needed. The method described here can be applied to study protein dynamics and function in vivo using ligands coupled to different fluorescent dyes. These ligands can enter the cell and specifically label proteins fused to HaloTag®. Several different protein fusions were made to demonstrate proper subcellular localization and protein migration. These ligands may also be rapidly switched, resulting in differential labeling of intracellular proteins (pulse-chase labeling). Through the ability to easily interchange ligands, the HaloTag® technology eliminates the need to make multiple constructs containing protein of interest fused to different functional tags.