Live Chat Support Software
800.227.0627

HaloTag fusion proteins and conjugates for cell imaging

HaloTag® fusion proteins and conjugates for cell imaging

By Klaus D. Linse 


The HaloTag® fusion protein technology can be used to design and prepare fusion proteins, fusion protein conjugates, and protein conjugates useful as tools for cell imaging.

 

Several approaches allow tethering of organic probes directly to specially designed reporter molecules. Many reporter proteins, such as the green fluorescent protein, are conjugated to affinity probes. Affinity probe can be a peptide, a protein or an oligonucleotide-based hybridization probe. However, any other affinity tag may be used as well. Often, proteins fused to selected tags are expressed in living cell. Many expressed proteins are found mainly in the inclusion bodies and require refolding before their use. Often, refolding is not successful. The HaloTag® fusion proteins are more soluble and, therefore, provide a means for the production of soluble recombinant proteins.

 

Typical applications for HaloTag fusion proteins and conjugates are:

 

1.      Affinity capture of various biomolecules;

2.      Chromatin immunoprecipitation;

3.      Fluorescent dye-based detection of biomolecules;

4.      Hydrophobic tagging induced protein degradation;

5.      Protein pull-downs;

6.      Protein purification for mammalian proteins;

7.      Protein purification of e. coli proteins or from other cell cultures;

8.      Protein solubilization during expression;

9.      Protein arrays;

10.    Protein-oligonucleotide conjugates for capture or detection probes;

11.    Quantum dot based detection of biomolecules;

12.    etc.

 

The HaloTag® fusion protein technology, developed by scientists at Promega Inc., offers a versatile tool for the study of molecular interaction within cells. To achieve optimal results the HaloTag® fusion protein technology allows for imaging of fixed or live cells. The key to the HaloTag® approach is the covalent tethering of organic probes directly to a specially designed reporting protein that can be expressed in living cells. The HaloTag® approach allows the introduction of genetically-encoded fusion proteins into living cells for imaging with the help of chemical conjugation probes. The model of a HaloTag® fusion protein is shown in figure 1.




Figure 1: Model of the HaloTag® protein. The binding pocket for covalent interaction with the HaloTag® ligands is shown to scale. (Source: © Promega Corporation. Reused with permission of Promega Corporation).

 

The HaloTag® reporter protein can be conjugated to other molecules such as receptor proteins, peptides or oligonucleotides. Various cloning strategies allows designing fusion proteins that may contain whole proteins, protein domains, protein binding motifs, or peptides as well as linkers and spacers. For example, the tobacco etch virus (TEV) protease cleavage site can be incorporated between the HaloTag® protein and the selected fusion protein (Figure 2). The incorporation of this cleavage site allows releasing the fusion protein from the tag. This is a very useful feature if the fusion protein needs to be purified. For the conjugation of specific probes, such as cyclic peptides or single-stranded and double-stranded oligonucleotides useful for RNAi experiments, chemical conjugation is needed. To achieve this, a correctly designed HaloTag® reporter protein containing a functional group accessible for conjugation reactions can be used.


Figure 2: Model of a HaloTag® fusion protein. The linker with the TEV site and the binding pocket for covalent interaction with the HaloTag® ligands is shown to scale. (Source: © Promega Corporation. Reused with permission of Promega Corporation).


Biomolecular imaging of living cells allows for the localization and quantification of biological events in cells. Using this technology as well as other similar approaches allows scientists to generate a more realistic view of the dynamics of cell metabolism and cell biology. Increasingly, it has become important to understand the functional roles of proteins, Peptides Synthesis, oligonucleotides such as various RNA molecules, carbohydrate structures, and other biological molecules in the cell and how they interact with each other within cells.


Improvements made in microscopy-based cell imaging technologies and instrumentation have enabled the study of molecules at the nanoscale level in more detail and higher resolution than ever before. Cell imaging of living cells using specifically labeled probes such as proteins, peptides or oligonucleotides can reveal detailed information about molecular functions, dynamics and their location of targeted biomolecules in cells. In the past decades, new innovative methods and technologies have been developed to enable specific labeling of diverse molecules for their use as molecular probes. A wide range of compounds with different optical properties and functionalities can be designed.


The HaloTag® methodology can be used for profiling of drugs and lead compounds by screening a wide array of cellular pathways. This approach allows for the identification of on-target and off-target activities of drugs in mammalian and human cells. Using this approach together with high-content, cell-based methods allow for drug discovery within living cells. The use of such methods is thought to allow for a better understanding of the nature of cell signaling, cellular networks, and drug effects influencing those networks. 


Fluorescent dyes and quantum dots conjugated to a chloroalkane group have already been used for fluorescence imaging of cells and cell compartments. This approach combines a genetically encoded tag, the HaloTag protein, with covalent labeling of the tag using ligands. Furthermore, the use of multicolor imaging of cellular proteins enables the imaging of different proteins in the same cell. This is possible if several different fusion tags are used in the same cell.


Liu et al. in 2013 reported the development of a method for targeting quantum dots (QD) to proteins in living cells. This QD targeting method is based on E. coli lipoic acid ligase (LplA) ligation of a haloalkane to a ligase acceptor peptide (LAP) fusion protein, followed by detection with HaloTag®-conjugated QDs. 


Figure 3: Schematic representation of HaloTag® -TMR ligand binding to the active site in the protein. Schematic representation of HaloTag® -TMR ligand binding to the active site in the protein. HaloTag® ligands with different functional groups are shown. Functional groups such as surfaces, e.g. beads, fluorescent dyes or reactive groups can be modified with the constant binding group, the chloroalkane ligand group. Depending on the nature of the functional group multiple functions supporting imaging, immobilization and other, can be added to a HaloTag® fusion protein. The final protein construct can be used in a number of in vitro and in vivo assay. (Source: © Promega Corporation. Reused with permission of Promega Corporation).


Furthermore, HaloTag® fusion proteins can be expressed and purified by affinity chromatography or by using a combination of chromatographic methods to achieve a highly pure protein. If the HaloTag® is fused to a target protein as a reversible tag the HaloTag® approach can be used to pull out proteins or protein complexes that bind to the selected fusion protein or probe from cells using affinity capture. One elegant approach to capture proteins and protein complexes is the use of biotinylated ligands together with solid supports such as streptavidin-coated particles or beads. If the HaloTag® is fused to a single- or double-stranded oligonucleotide an affinity capture probe can be designed, for example, to pull-down miRNA and miRNA-protein complexes.


For protein expression, HaloTag Vectors are used. Several vectors for encoding of fusion and tag proteins and modified ligands have now been developed. Both, vectors and HaloTag® Ligands are available from Promega. 


A general process for cell-based applications includes the following steps:


(1)    Make a vector encoding a fusion of the HaloTag® protein to a protein,
         a protein-domain, or peptide sequence of interest. A cleavage site or
         linker peptide may also be included;

(2)    Express the fusion chimera in cells;

(3)    Label the cells with the HaloTag ligand;

(4)    Image the sample, either as live or fixed cells.

(5)    If desired, purify the protein from cell cultures.

HaloTag® protein engineering


The HaloTag® protein is an engineered, catalytically inactive derivative of a bacterial hydrolase. A hydrolase acts on halide bonds in carbon-halide compounds. The native hydrolase has a molecular weight of approximately 33 kDa and exists as a monomer. The enzyme cleaves carbon-halogen bonds in aliphatic halogenated compounds. The reaction involves a hydrolytic triad at the active site of the protein.

 

R-Cl + Enzyme + HOH  -> R-Enzyme + Cl- +HOH -> R-OH + H+ + Cl-


The native enzyme catalyzes the reaction by forming an enzyme-substrate complex. The reaction involves a nucleophilic attack involving Asp106 to form an ester intermediate with the halide group in the catalytic center. His272 activates H2O that hydrolyzes the intermediate. Finally, the product is released from the catalytic center. A His272Phe substitution in the protein impairs the hydrolysis step. However, a covalent bond between protein and ligand can be formed. The ligand can contain a functional reporter group useful for imaging applications. Further optimization of the amino acid sequence of the protein provided a better access to the active site by different modified ligands. This resulted in a dramatic increase in the ligand binding rate by several thousand-fold. An almost immediate binding of the HaloTag® TMR Ligand binding to GST-HaloTag® fusion protein is reported.


Haloalkane Dehalogenases

 

Haloalkane dehalogenases are known to catalyze the hydrolytic cleavage of carbon-halogen bonds. This is a key step to mineralization of many pollutants. The molecular model of the HaloTag 7 haloalkane dehalogenase at a high resolution is illustrated in figure 4. The sequence annotated with secondary structure information is shown as well.



Figure 4: Model of HaloTag 7 haloalkane dehalogenase.

Lahoda et al. in 2014 reported the design of a haloalkane dehalogenase variant Dha A31 that showed increased catalytic activity toward 1,2,3-trichloroporpane (TCP), and important toxic pollutant. This enzyme could be used for the bio-remediation of TCP. The model of this structure is shown in figure 5.

Figure 5: Molecular models of haloalkane dehalogenase variant Dha A31.

 

Figure 6: Molecular models of a haloalkane dehalogenase complexed with halid ions.

 

Reference

 

HaloTag® Technology: Focus on Imaging. Technical Manual. Promega. TM260.

http://www.promega.com

http://www.promega.com/resources/vector-sequences/halotag-vectors/


Lahoda
, M.,Mesters, J.R., Stsiapanava, A., Chaloupkova, R., Kuty, M., Damborsky, J., Kuta Smatanova, I.; Crystallographic analysis of 1,2,3-trichloropropane biodegradation by the haloalkane dehalogenase DhaA31. (2014) Acta Crystallogr.,Sect.D 70: 209-217 PubMed: 24531456 Search on PubMed DOI: 10.1107 / S1399004713026254  Primary Citation of Related Structures: 3RK4, 4FWB, 4HZG.

Daniel S. Liu
, William S. Phipps, Ken H. Loh, Mark Howarth, and Alice Y. Ting; Quantum Dot Targeting with Lipoic Acid Ligase and HaloTag for Single Molecule Imaging on Living Cells. ACS Nano. 2012 December 21; 6(12): 11080–11087. doi:10.1021/nn304793z.


Scott N. Peterson and Keehwan Kwon;
The HaloTag: Improving Soluble Expression and Applications in Protein Functional Analysis. Current Chemical Genomics, 2012, 6, (Suppl 1-M2) 8-17.


Sun
et al. (2015), HaloTag is an effective expression and solubilisation fusion partner for a range of fibroblast growth factors. PeerJ 3:e1060; DOI 10.7717/peerj.1060


Urh
, M. and Rosenberg, M.; HaloTag, a Platform Technology for Protein Analysis. Current Chemical Genomics, 2012, 6, (suppl 1-M8) 72-78.

-.-