While GST and 6XHis tags are useful for isolating proteins expressed in bacterial cells, there are tagging options that are compatible with both prokaryotes (e.g., E. coli) and eukaroytes (e.g., mammalian cells). The 34kDa HaloTag® protein offers this expression system flexibility. In contrast to other tags that use noncovalent interactions to purify proteins, the basis of HaloTag® technology is the covalent binding between HaloTag® protein and its ligand. Because of the strength of the covalent bond, you can wash your tagged protein under stringent conditions and basically eliminate any nonspecific protein background. Worried about the size of the tag affecting the function of your protein? No problem! Just use TEV protease to cleave your protein from HaloTag and the purified, untagged protein is ready to use in downstream analysis. Learn more about HaloTag® Technology here.
One of the challenges of expressing proteins in bacterial cells is solubility. Eukaryotic proteins are not methylated and lack other post-translational modifications when synthesized in E. coli, making them prone to forming inclusion bodies. Fusion tags like that of HaloTag® protein can make recombinant proteins more soluble and even enhance expression in bacteria, making your protein of interest easier to purify. By expressing your HaloTag®-protein fusion in mammalian cells, post-translational modifications will be present, more closely reflecting how the protein will function under cellular conditions. Using an immobilized matrix like resin coated in HaloTag® ligand, you can purify only proteins with HaloTag present or use in pull-down assays for discovering what protein or proteins bind to your protein of interest. In this way, you can better understand protein interactions.
But the applications don’t stop with protein:protein interactions and protein purification. If you want to explore what DNA sequence your protein binds, HaloTag® technology can help decipher the protein:DNA interaction with the HaloCHIP™ System. Want to use immunochemistry? We have an antibody for that. We have an antibody for that. The Anti-HaloTag® pAb is a good place to start.
Interested in cell imaging? You will need to pick the right fluorescent ligand (e.g., HaloTag® Ligands or Janelia Fluor® HaloTag® Ligands). With your protein fused to HaloTag® protein and fluorescent HaloTag® ligands, the fluorescent dyes provide labeling or detection options for studying protein:protein interactions, protein localization, protein trafficking and turnover, and cell counting using FACS. The brightness and stability of the Janelia Fluor® HaloTag® Ligands means they are especially useful for intracellular imaging, including single-molecule imaging and tracking in live or fixed cells, confocal fluorescent imaging and super resolution microscopy techniques including dSTORM.
Not seeing the ligand you need for your project? Create virtually any HaloTag® Ligand you can imagine using the HaloTag® Ligand Building Blocks. The chloroalkane group to which HaloTag® protein covalently bonds can be applied to any compound or surface with a compatible chemical group.
To get started with HaloTag® technology, clone the coding region for your protein of interest in traditional or Flexi® cloning vectors to add HaloTag at the N or C terminus. Alternatively, you can search for any of the thousands of prebuilt HaloTag® clones available from Kazusa. Once you have your clone, you can use it for any protein analysis application. Depending on what you want to do next, you may need a purification system (e.g., HaloTag® Mammalian Protein Purification System or HaloTag® Protein Purification System) or a pull-down assay (e.g., HaloTag® Mammalian Pull-Down Systems). There are bright fluorescent HaloTag® Ligands or Janelia Fluor® HaloTag® Ligands to use for imaging studies, the Anti-HaloTag® pAb for immunological detection and the NanoBRET™ PPI Systems (for protein:protein interactions) and HaloCHIP™ System (for protein:DNA interactions) to examine protein interations.