Development of a Dehalogenase-Based Protein Fusion Tag Capable of Rapid, Selective and Covalent Attachment to Customizable Ligands

Abstract

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins.

CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells

“…The Halo tag can be labeled efficiently and covalently by Halo ligands conjugated to a variety of organic fluorescent dyes for different imaging purposes (10). We purified recombinant dCas9 fusion protein expressed in Escherichia coli and labeled the protein with Halo ligands conjugated with Janelia Fluor 646 (JF646) (11).…”

A Bidirectional System for the Dynamic Small Molecule Control of Intracellular Fusion Proteins

“…To increase its stability, 22 mutations have previously been introduced to create the more stable HaloTag7 protein for recombinant protein work 15; 16 . We hypothesized that these mutations would also provide increased stability in mammalian cells.…”

Signal Transduction: From the Atomic Age to the Post-Genomic Era

“…Similarly, mutagenesis of small single-chain enzymes has yielded variants that self-label covalently with a fluorophore substituent in the course of one catalytic turnover when provided with an appropriate cell-permeable, dye-derivatized substrate. This approach produced the so-called SNAP (Corrêa et al 2013), CLIP (Gautier et al 2008), and Halo (Encell et al 2012) tags. It is important to emphasize, however, that studies tracking hybrid proteins must be confirmed by independent approaches (e.g., genetic complementation) that show they retain biological function; sadly, such constructs are often used and conclusions drawn without this necessary validation.…”