No single fusion tag served expression, solubility and purification for difficult human proteins in E. coli, so tags were often combined. This paper tested HaloTag7 on 23 human proteins, of which 74% were soluble versus 52% for MBP, 39% for GST and 22% for His6 Tag.
In E. coli KRX, N-terminal HaloTag7 fusions keep 74% of a 23-protein panel of human proteins (9 to 155 kDa, chosen for poor expression without a tag) soluble and detectable by SDS-PAGE, compared with 52% for N-terminal MBP, 39% for GST and 22% for His6 Tag fusions. The six proteins not detected in soluble form as HaloTag7 fusions were not solubilized by any of the other three tags.
02
Soluble expression of N-terminal fusions to human proteins in E. coli KRX decreases with increasing target protein size for HaloTag7, MBP, GST and His6 Tag alike, but the decline is smaller for HaloTag7. Among targets of 60 kDa or larger, HaloTag7 gives soluble expression for 37% of proteins, compared with 12.5% for MBP and 0% for both GST and His6 Tag.
03
Covalent capture of HaloTag7 fusions on HaloLink resin, followed by TEV protease release of the target, purified five of six test human proteins expressed in E. coli KRX, compared with three of six for GST and for MBP and one of six for His6 Tag (purified from 1 ml cultures). The 78 kDa protein kinase C gamma (PRKCG), which had no detectable soluble expression as any of the four fusions, was purified only as a HaloTag7 fusion.
04
Human proteins purified from E. coli through HaloTag7 capture on HaloLink resin and TEV release reach at least 95% purity, compared with 62 to 84% for GST and 30 to 60% for MBP released from their resins by TEV protease. The lower GST and MBP purity comes mainly from free tag released from the resin during proteolytic cleavage, whereas HaloTag7 stays covalently bound to the resin.
05
HaloTag7 binds a chloroalkane ligand covalently and essentially irreversibly, so a HaloTag7 fusion on HaloLink resin cannot be eluted and is instead released by cleavage at an optimized TEV recognition site in the interconnecting polypeptide. The released target carries six vector-derived N-terminal residues (SDNAIA), and the tag remains on the resin, which removes free tag and uncleaved fusion from the product.
06
Human proteins purified from E. coli after TEV release from HaloTag7, GST or MBP resins, or eluted from His6 Tag resin with imidazole, show gel filtration molecular weights in good agreement with theoretical values, typically within 10% (20% for the 19.7 kDa TCTP), and elute as non-aggregating monomers. The exception is the 9.7 kDa small muscular protein SMPX, which separates into multiple peaks with HaloTag7 (calculated 10, 32 and 67 kDa, a monomer, trimer and hexamer) and into trimer and hexamer peaks with GST.
07
The mouse Id protein and the human protein kinase A catalytic subunit (PKAc), expressed as N-terminal fusions in E. coli KRX and purified from 50 ml cultures, have higher specific activity when purified through HaloTag7 with TEV release than through the other tags. After correcting for purity, Id purified via HaloTag7 binds MyoD 50 to 60% more actively, and PKAc phosphorylates Kemptide 15 to 30% more actively, than the same proteins purified via GST, MBP or His6 Tag.
08
PKAc purified as an uncleaved His6 Tag fusion from E. coli has significantly lower specific activity than PKAc purified from GST, MBP or HaloTag7 fusions after tag removal. The possible causes named are interference by the retained His6 Tag and residual imidazole not removed by dialysis.
09
Labeling a HaloTag7 fusion in E. coli lysate with 1 uM HaloTag TMR ligand for 30 min forms a covalent bond that survives boiling in SDS loading buffer (2 min at 95 C) and SDS-PAGE, so labeled fusions are detected by in-gel fluorescence. All six test HaloTag7 fusions were detected, including PRKCG, which was not visible by SimplyBlue staining.
10
Labeling of HaloTag7 fusion proteins with the fluorescent chloroalkane ligand occurs at a 1:1 stoichiometry, so fusion protein expression in a lysate can be quantified from an in-gel linear calibration curve of serial dilutions of purified, TMR-labeled HaloTag7 (34 kDa) and GST:HaloTag7 (60 kDa).
11
In-gel fluorescence of TMR-labeled HaloTag7 fusions detects unintended truncations within the polypeptide linker region of the fusion that SimplyBlue staining does not reveal.
12
HaloTag7 is a 34 kDa monomeric protein, a catalytically inactive derivative of DhaA, a haloalkane dehalogenase from Rhodococcus, engineered by rational design and molecular evolution for rapid covalent attachment to chloroalkane ligands and for enhanced expression and solubility when fused to a partner. Relative to the parental dehalogenase it has increased stability and negative charge, and the proposed explanation for its solubility effect is that this stability promotes folding of fusion partners and the uniform negative surface charge reduces aggregation.
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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 His6 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.
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