HaloTag7: A genetically engineered tag that enhances bacterial expression of soluble proteins and improves protein purification
ABSTRACT 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.
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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 01/2014; 5:63. DOI:10.3389/fmicb.2014.00063
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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
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ABSTRACT: 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.Current Chemical Genomics 09/2012; 6(1):8-17. DOI:10.2174/1875397301206010008