HaloTag7: A genetically engineered tag that enhances bacterial expression of soluble proteins and improves protein purification

Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA
Protein Expression and Purification (Impact Factor: 1.7). 11/2009; 68(1):110-120. DOI: 10.1016/j.pep.2009.05.010
Source: PubMed


Over-expression and purification of soluble and functional proteins remain critical challenges for many aspects of biomolecular research. To address this, we have developed a novel protein tag, HaloTag7, engineered to enhance expression and solubility of recombinant proteins and to provide efficient protein purification coupled with tag removal. HaloTag7 was designed to bind rapidly and covalently with a unique synthetic linker to achieve an essentially irreversible attachment. The synthetic linker may be attached to a variety of entities such as fluorescent dyes and solid supports, permitting labeling of fusion proteins in cell lysates for expression screening, and efficient capture of fusion proteins onto a purification resin. The combination of covalent capture with rapid binding kinetics overcomes the equilibrium-based limitations associated with traditional affinity tags and enables efficient capture even at low expression levels. Following immobilization on the resin, the protein of interest is released by cleavage at an optimized TEV protease recognition site, leaving HaloTag7 bound to the resin and pure protein in solution. Evaluation of HaloTag7 for expression of 23 human proteins in Escherichia coli relative to MBP, GST and His6Tag revealed that 74% of the proteins were produced in soluble form when fused to HaloTag7 compared to 52%, 39% and 22%, respectively, for the other tags. Using a subset of the test panel, more proteins fused to HaloTag7 were successfully purified than with the other tags, and these proteins were of higher yield and purity.

1 Follower
30 Reads
  • Source
    • "MBP and GST bind to their substrates non-covalently. On the contrary, the HaloTag7 (Promega) is based on the covalent capture of the tag to the resin, making the system fast and highly specific (Ohana et al., 2009). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field.
    Frontiers in Microbiology 04/2014; 5:172. DOI:10.3389/fmicb.2014.00172 · 3.99 Impact Factor
  • Source
    • "The use of fusion partners was an important turning point for the E. coli host system: fusion tags promote or increase protein solubility, help on protein purification and can also be used to increase protein's immunogenicity. Traditional fusion systems like MBP, GST, NusA, or Trx have constantly been challenged and complemented by novel fusion solutions such as the SUMO tag (Butt et al., 2005; Marblestone et al., 2006), the HaloTag (Ohana et al., 2009), the SNUT tag (Caswell et al., 2010), and the expressivity tag (Hansted et al., 2011), among others. More recently, a novel and unique fusion system for simple and inexpensive soluble protein overproduction and purification in E. coli was developed and studied: the Fh8 tag (Costa, 2013). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Proteins are now widely produced in diverse microbial cell factories. The Escherichia coli is still the dominant host for recombinant protein production but, as a bacterial cell, it also has its issues: the aggregation of foreign proteins into insoluble inclusion bodies is perhaps the main limiting factor of the E. coli expression system. Conversely, E. coli benefits of cost, ease of use and scale make it essential to design new approaches directed for improved recombinant protein production in this host cell. With the aid of genetic and protein engineering novel tailored-made strategies can be designed to suit user or process requirements. Gene fusion technology has been widely used for the improvement of soluble protein production and/or purification in E. coli, and for increasing peptide's immunogenicity as well. New fusion partners are constantly emerging and complementing the traditional solutions, as for instance, the Fh8 fusion tag that has been recently studied and ranked among the best solubility enhancer partners. In this review, we provide an overview of current strategies to improve recombinant protein production in E. coli, including the key factors for successful protein production, highlighting soluble protein production, and a comprehensive summary of the latest available and traditionally used gene fusion technologies. A special emphasis is given to the recently discovered Fh8 fusion system that can be used for soluble protein production, purification, and immunogenicity in E. coli. The number of existing fusion tags will probably increase in the next few years, and efforts should be taken to better understand how fusion tags act in E. coli. This knowledge will undoubtedly drive the development of new tailored-made tools for protein production in this bacterial system.
    Frontiers in Microbiology 02/2014; 5:63. DOI:10.3389/fmicb.2014.00063 · 3.99 Impact Factor
  • Source
    • "Furthermore , while the level of expression per unit of culture density, and the solubility of recombinant proteins were comparable for both IPTG-induced and auto-induced cultures, higher culture densities as well as a higher content of target protein were reached under conditions of auto-induction [15]. To our knowledge, there is only one report on successful purification of E. coli-expressed, soluble proteins using the HaloTag technology [9]. In that case, expression was performed in E. coli strain KRX by auto-induction. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Expression of milligram quantities of functional, stable G protein-coupled receptors (GPCR) for high-resolution structural studies remains a challenging task. The goal of this work was to evaluate the usefulness of the HaloTag system (Promega) for expression and purification of the human cannabinoid receptor CB2, an important target for development of drugs for treatment of immune disorders, inflammation, and pain. Here we investigated expression in Escherichia coli cells of the integral membrane receptor CB2 as a fusion with the 34 kDa HaloTag at N- or C-terminal location, either in the presence or in the absence of the N-terminal maltose-binding protein (MBP). The CB2 was flanked at both ends by the tobacco etch virus (TEV) protease cleavage sites to allow for subsequent removal of expression partners. Expression by induction with either IPTG (in E. coli BL21(DE3) cell cultures) or by auto-induction (in E. coli KRX cells) were compared. While the N-terminal location of the HaloTag resulted in high levels of expression of the fusion CB2, the recombinant receptor was not functional. However, when the HaloTag was placed in the C-terminal location, a fully active receptor was produced irrespective of induction method or bacterial strain used. For purification, the fusion protein was captured onto HaloLink resin in the presence of detergents. Treatment with specific TEV protease released the CB2 upon washing. To our knowledge, this study represents the first example of expression, surface immobilization and purification of a functional GPCR using HaloTag technology.
    Protein Expression and Purification 03/2013; 89(1). DOI:10.1016/j.pep.2013.02.011 · 1.70 Impact Factor
Show more