title: 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 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.
authors.affiliation: Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA. rachel.ohana@promega.com
authors.forename: Rachel Friedman
authors.initials: RF
authors.lastname: Ohana
authors.forename: Lance P
authors.initials: LP
authors.lastname: Encell
authors.forename: Kate
authors.initials: K
authors.lastname: Zhao
authors.forename: Dan
authors.initials: D
authors.lastname: Simpson
authors.forename: Michael R
authors.initials: MR
authors.lastname: Slater
authors.forename: Marjeta
authors.initials: M
authors.lastname: Urh
authors.forename: Keith V
authors.initials: KV
authors.lastname: Wood
mesh_terms: D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D010865:Pilot Projects; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012995:Solubility
chemical_list: D011993:Recombinant Fusion Proteins