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.

Introduction

Recombinant DNA technologies have greatly expanded our access to the broad diversity of proteins represented in living organisms, 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 structures and interactions within the host organism, success with these techniques often remains frustratingly elusive. Successful purification 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 documented protocols [1], [2]. However, over-expression of heterologous proteins in E. coli, particularly human proteins, often yields inadequate levels of soluble protein [1], [2], [3], [4].

One approach for overcoming this limitation is to optimize expression conditions such as temperature, growth media, induction parameters, promoters and E. coli expression strain [1], [5]. Systematic screening of such variables can be simplified by using reporter fusion tags such as GFP¹ [6], [7] or S-tag [8]. Another common 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 different 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 adaptable to all target proteins. Many affinity tags have been developed, ranging in size from a few amino acids to entire proteins, that are capable of selective interaction with their corresponding ligands coupled to a chromatography matrix [17]. His₆Tag [18], [19] is most widely used due to its small size and ability to frequently provide sufficient protein yield and purity for many applications. Other common affinity tags include GST and MBP, which are often favored for their additional ability to enhance protein solubility [3], [4], [20].

Although appending a fusion tag onto a protein of interest can improve expression and purification, the tag can also interfere with protein structure or function [13], [21], [22]. Consequently, it is commonly recommended to remove the tag after purification [18], [21], [22]. Tag removal can be performed by proteolytic cleavage at a defined sequence in the interconnecting polypeptide, i.e. linker separating the tag and the target protein [18], [23], [24], [25]. This approach can be problematic due to non-specific or inefficient cleavage or loss of protein stability and solubility following tag removal [25], [26], [27]. Furthermore, this step often requires additional effort to separate the free target protein from the affinity tag and the protease.

While a variety of fusion tags is available to facilitate aspects of protein expression, solubility, detection, or purification, most tags are lacking or inefficient in some of these features. Many proteins are poorly expressed with available solubility tags, or are difficult to purify with existing affinity technologies due to low binding onto the purification matrix [25], [26], [28], [29]. These shortcomings are addressed by a new tag, HaloTag7, designed to support efficient expression of soluble protein and bind rapidly and covalently to a unique synthetic ligand. HaloTag7 is a catalytically inactive derivative of DhaA, a bacterial haloalkane dehalogenase from Rhodococcus [30] present only among selected microbial groups. This 34 kDa monomeric protein was engineered through rational design and molecular evolution to rapidly form a covalent attachment to synthetic chloroalkane ligands [31], [32], and to provide enhanced expression and solubility when fused to a protein partner.

The synthetic ligands comprise a chloroalkane linker attached to a variety of functional groups including fluorophores, affinity handles and solid supports. These features enable both fluorescent labeling of fusion proteins in cell lysate for expression screening and irreversible capture of fusion proteins onto a purification matrix. The rapid, specific, and covalent capture offered by HaloTag7 overcomes the inherent limitation of affinity tags by effectively eliminating protein loss associated with equilibrium-based binding. This feature is especially important for purification of poorly expressed proteins. Following immobilization on the purification matrix, the target protein can be released by cleavage at an optimized TEV protease recognition site contained within the interconnecting polypeptide separating HaloTag7 and the fusion partner. The HaloTag7-based protein purification method yields highly pure proteins in solution while the fusion tag remains covalently attached to the matrix, eliminating contamination by free tag or un-cleaved fusion protein.

To demonstrate the efficacy of HaloTag7 for protein expression and purification with E. coli, we compared its performance to the commonly used affinity tags, GST, MBP, and His₆Tag (see Table 1 for tags characteristics). We chose GST and MBP as they are used in a manner similar to HaloTag7; both promote expression of soluble protein in E. coli and both provide a means for protein purification. Although His₆Tag does not assist in protein expression or solubilization, this tag was also chosen because of its widespread use for protein purification. The relative performance of these tags was evaluated using a panel of cDNA clones encoding 23 human proteins that are difficult to express in E. coli [33]. The set of proteins ranges broadly in both size (∼9–155 kDa) and function (e.g. kinases, membrane proteins and transcription factors). Our results showed that HaloTag7 delivered superior performance for protein expression, solubility, purification yield and purity. Furthermore, using two additional model proteins, we found that HaloTag7 produced proteins with higher specific activity.