HaloTag Research Articles

The HaloTag: Improving Soluble Expression and Applications in Protein Functional Analysis

Technological and methodological advances have been critical for the rapidly evolving field of proteomics. The development of fusion tag systems is essential for purification and analysis of recombinant proteins. The HaloTag is a 34 KDa monomeric protein derived from a bacterial haloalkane dehalogenase. The majority of fusion tags in use today utilize a reversible binding interaction with a specific ligand. The HaloTag system is unique in that it forms a covalent linkage to its chloroalkane ligand. This linkage permits attachment of the HaloTag to a variety of functional reporters, which can be used to label and immobilize recombinant proteins. The success rate for HaloTag expression of soluble proteins is very high and comparable to maltose binding protein (MBP) tag. Furthermore, cleavage of the HaloTag does not result in protein insolubility that often is observed with the MBP tag. In the present report, we describe applications of the HaloTag system in our ongoing investigation of protein-protein interactions of the Y. pestis Type 3 secretion system on a custom protein microarray. We also describe the utilization of affinity purification/mass spectroscopy (AP/MS) to evaluate the utility of the Halo Tag system to characterize DNA binding activity and protein specificity.

Establishment of a Novel Fluorescence-Based Method to Evaluate Chaperone-Mediated Autophagy in a Single Neuron

BackgroundChaperone-mediated autophagy (CMA) is a selective autophagy-lysosome protein degradation pathway. The role of CMA in normal neuronal functions and in neural disease pathogenesis remains unclear, in part because there is no available method to monitor CMA activity at the single-cell level.Methodology/Principal FindingsWe sought to establish a single-cell monitoring method by visualizing translocation of CMA substrates from the cytosol to lysosomes using the HaloTag (HT) system. GAPDH, a CMA substrate, was fused to HT (GAPDH-HT); this protein accumulated in the lysosomes of HeLa cells and cultured cerebellar Purkinje cells (PCs) after labeling with fluorescent dye-conjugated HT ligand. Lysosomal accumulation was enhanced by treatments that activate CMA and prevented by siRNA-mediated knockdown of LAMP2A, a lysosomal receptor for CMA, and by treatments that inactivate CMA. These results suggest that lysosomal accumulation of GAPDH-HT reflects CMA activity. Using this method, we revealed that mutant γPKC, which causes spinocerebellar ataxia type 14, decreased CMA activity in cultured PCs.Conclusion/SignificanceIn the present study, we established a novel fluorescent-based method to evaluate CMA activity in a single neuron. This novel method should be useful and valuable for evaluating the role of CMA in various neuronal functions and neural disease pathogenesis.

The use of HaloTag-based technology in flow and laser scanning cytometry analysis of live and fixed cells

BackgroundCombining the technologies of protein tag labeling and optical microscopy allows sensitive analysis of protein function in cells.FindingsHere, we describe development of applications using protein tag technology (HaloTag (HT)-based) for flow and laser scanning cytometry (LSC). Cell lines, expressing recombinant surface β1-integrin-HT and HT-p65 fusion protein, and a CD4 T cell line (Jurkat) infected with human immunodeficiency virus type 1 (HIV-1) reporter virus expressing the unfused HT (HIV-1Lai-Halo), were stained with different HT ligands and successfully detected by flow cytometers equipped with 488 and 561 nm lasers as well as a laser scanning cytometer (equipped with 488 and 405 nm lasers) alone or combined with cell cycle and viability markers.ConclusionsUse of HT technology for cytometric applications has advantages over its use in microscopy as it allows for the statistical measurement of protein expression levels in individual cells within a heterogeneous cell population in combination with cell cycle analysis. Another advantage is the ability of the HaloTag to withstand long fixation and high concentration of fixative, which can be useful in research of infectious agents like HIV and/or mycobacteria.

Massive Endocytosis (MEND) Activated By Ca and Polyamines in Fibroblasts and Cardiac Myocytes: Optical Studies of Membrane and Na/Ca Exchanger (NCX1) Internalization, The Possible Role of Ca-activated Transglutaminase in MEND, and The Possible Function of MEND In Cardiac Myocytes

When cytoplasmic solutions contain physiological concentrations of polyamines (1 mM spermine or spermidine), large Ca transients can cause massive endocytosis in fibroblasts and cardiac myoyctes within 2 to 6 seconds. The magnitudes of responses (30 to 70% of cell surface) are confirmed by both capacitance and FM dye measurements. Using cardiac Na/Ca exchanger (NCX1) fusions with an extracellular Halo tag or an extracellular Phluorin tag, we confirm that NCX1 can be internalized during MEND. We next describe experiments to test candidates for the Ca sensor in MEND. Calmodulin-dependent processes have been largely eliminated by inhibitor studies. Ca-activated transglutaminase activity appears to be an interesting candidate with the potential that critical signaling proteins are activated by polyamination. MEND is inhibited by the transglutaminase blocker, cystamine, and by glutamine-containing peptide substrates of transglutaminase. We also describe that MEND-like processes can be activated in cardiac myoytes during cell isolation protocols. In myocytes from young rodents, NCX1 can be lost ‘biochemically’ and ‘functionally’ from the cell surface within 30 min to 1 h, and this loss can be largely blocked by cystamine. Furthermore, HRP labeling progresses rapidly through vesicle, multivesicular, and lysosome stages during this same time period after isolation.