HaloTag7: A genetically engineered tag that enhances bacterial expression of soluble proteins and improves protein purification Rachel Friedman Ohana * , Lance P. Encell, Kate Zhao, Dan Simpson, Michael R. Slater Marjeta Urh, Keith V. Wood Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA article info Article history: Received 12 May 2009 and in revised form 16 May 2009 Available online 21 May 2009 Keywords: HaloTag7 Protein expression Protein solubility Protein purification Fusion tag Protein labeling Enhanced expression in E. coli HaloTag 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, engi- neered 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 purifica- tion 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 His 6 Tag 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. Ó 2009 Elsevier Inc. All rights reserved. Introduction Recombinant DNA technologies have greatly expanded our ac- cess to the broad diversity of proteins represented in living organ- isms, and potentially to an even broader range of mutant proteins not found in nature. Accordingly, the expression and purification of recombinant proteins has become fundamental to many aspects of life science research. Yet, owing to complexities in protein struc- tures and interactions within the host organism, success with these techniques often remains frustratingly elusive. Successful purifica- tion of functional proteins generally requires efficient expression of these proteins in soluble form followed by their separation from the highly complex crude lysate of the host. The most frequently used host for protein expression is Escherichia coli due to its ease of use, rapid cell growth, low cost of culturing and well docu- mented protocols [1,2]. However, over-expression of heterologous proteins in E. coli, particularly human proteins, often yields inade- quate levels of soluble protein [1–4]. One approach for overcoming this limitation is to optimize expression conditions such as temperature, growth media, induc- tion parameters, promoters and E. coli expression strain [1,5]. Systematic screening of such variables can be simplified by using reporter fusion tags such as GFP 1 [6,7] or S-tag [8]. Another com- mon strategy is to use solubility fusion tags for boosting expression of soluble protein, presumably by promoting proper folding of the fusion partner and suppressing proteolysis [1,9]. A variety of differ- ent solubility tags are available, yet not all are equally efficient as solubility enhancers. The most commonly used include GST [10,11], TRX [12], MBP [13,14] and NusA [15,16]. Once adequate expression of soluble protein is achieved, the next step is to purify the target protein from the biological mixture. Affinity tags are widely used to simplify the purification process and to provide a generic method that is straightforward and adapt- able to all target proteins. Many affinity tags have been developed, 1046-5928/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2009.05.010 * Corresponding author. Fax: +1 608 277 2601. E-mail address: rachel.ohana@promega.com (R.F. Ohana). 1 Abbreviations used: amp, ampicilin; DTT, diothiothreitol; Escherichia coli, E. coli; EDTA, ethylenediaminetetraacetic acid; GFP, green fluorescent protein; GST, gluta- thione-S-transferase; kan, kanamycin; LB, Luria broth; MBP, maltose binding protein; NusA; N utilization substance A; MW, molecular weight; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; TRX, thioredoxin; TEV, tobacco etch virus; TMR, tetramethylrhodamine. Protein Expression and Purification 68 (2009) 110–120 Contents lists available at ScienceDirect Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep