Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Abstract

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast. In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1. These examples demonstrate the power of this technology in enabling the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics.

HaloTag Technology: A Versatile Platform for Biomedical Applications

“… 56 For example, interactions of bromodomain protein (BRD4) and histone deacetylase (HDAC1) with additional proteins were examined with the assistance of HaloTag. 57 In this pull-down assay, the bait protein linked to HaloTag formed standard protein–protein interactions within the cell. Next, cells were lysed to release the protein complexes and analyzed with liquid chromatography–mass spectrometry (LC–MS) to determine interacting proteins (Figure 4 A).…”

“…Next, cells were lysed to release the protein complexes and analyzed with liquid chromatography–mass spectrometry (LC–MS) to determine interacting proteins (Figure 4 A). 57 Pull-down assays are relatively common for protein analysis, as they provide details regarding complex protein interactions and possibly novel interactions. Additional studies evaluating the use of HaloTag for the extraction and purification of protein complexes using pull-down assays have been performed in both bacteria and mammalian cells.…”

“…Pure proteins can be eluted using a detergent (e.g., SDS), or protein complexes attached the bait protein can be eluted using TEV cleavage. Reprinted with permission from ref ( 57 ). Copyright 2014 JoVE.…”

Nanoscale gizmos – the novel fluorescent probes for monitoring protein activity

“…Halotags have been applied to monitor protein-protein and protein-DNA interactions. They have been used to monitor the interaction of bromodomain protein (BRD4) and histone deacetylase (HDAC1) along with other additional proteins [57]. Halotag has been adapted for the investigation of epidermal growth factor receptor Rasextracellular signal regulated kinase (ERK) mitogen activated protein (MAP) kinase pathway in living cells.…”

Targeted analysis of recombinant NF kappa B (RelA/p65) by denaturing and native top down mass spectrometry

“…Here, we utilized previously developed HaloTag fusion protein technology[20, 21] to isolate and purify p65 prior to targeted proteoform analysis by top down mass spectrometry; HaloTag is a modified bacterial dehalogenase enzyme that enables covalent capture and fluorescent labeling of recombinant proteins. With the advantages of efficient protein yield in non-denaturing conditions afforded by HaloTag purification, we assessed the different MS 1 strategies for measuring the intact mass of the 62 kDa p65 target, including denaturing LC-Orbitrap Fourier-transform mass spectrometry (FTMS) and native direct infusion on an Orbitrap FTMS, and finally obtained isotopic resolution by a 12 Tesla Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer.…”