While GST and 6XHis tags are useful for isolating proteins expressed in bacterial cells, there are tagging options that are compatible with both E. coli and 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.
One of challenges of expressing proteins in bacterial cells is solubility. Eukaroytic 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. What to use immunochemistry? We have an antibody for that. Interested in cell imaging? With your protein fused to HaloTag® protein and fluorescent HaloTag® ligands, you can find where your protein is localized, if it is trafficked and how quickly it turns over. There are even ligands for super resolution microscopy and FACS. Learn more about HaloTag® Technology here.
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). For imaging studies, you will need to pick the right fluorescent ligand (e.g., HaloTag® Ligands or Janelia Fluor® HaloTag® Ligands). If you are interested in immunological studies, the Anti-HaloTag® pAb would help. For protein interactions, there is the NanoBRET™ PPI Systems for protein:protein interaction studies and HaloCHIP™ System for examining protein:DNA interactions.
Figure 1. This illustration shows how HaloTag® protein is fused to a protein of interest and the covalent binding between HaloTag and its ligand.