HaloTag-based purification of functional human kinases from mammalian cells.
Although cultured mammalian cells are preferred for producing functional mammalian proteins with appropriate post-translational modifications, purification of recombinant proteins is frequently hampered by low expression. We have addressed this by creating a new method configured specifically for mammalian cell culture that provides rapid detection and efficient purification. This approach is based on HaloTag, a protein fusion tag designed to bind rapidly, selectively and covalently to a series of synthetic ligands that can carry a variety of functional groups, including fluorescent dyes for detection or solid supports for purification. Since the binding of HaloTag to the HaloLink resin is essentially irreversible, it overcomes the equilibrium-based binding limitations associated with affinity tags and enables efficient capture and purification of target protein, even at low expression levels. The target protein is released from the HaloLink resin by specific cleavage using a TEV protease fused to HaloTag (HaloTEV), leaving both HaloTag and HaloTEV permanently attached to the resin and highly pure, tag-free protein in solution. HaloTag fluorescent ligands enable fluorescent labeling of HaloTag fusion proteins, providing a convenient way to monitor expression, and thus facilitate the identification of optimal transient transfection conditions as well as the selection of high expression stable cell lines. The capabilities of this method have been demonstrated by the efficient purification of five functional human kinases from HEK293T cells. In addition, when purifications using FLAG, 3xFLAG, His(6)Tag and HaloTag were performed in parallel, HaloTag was shown to provide significantly higher yields, purity and overall recovery of the expressed proteins.
Promega Corporation, Madison, WI 53711, USA. rachel.ohana@promega.com
Rachel Friedman
RF
Ohana
Robin
R
Hurst
Jolanta
J
Vidugiriene
Michael R
MR
Slater
Keith V
KV
Wood
Marjeta
M
Urh
D015153:Blotting, Western; D018929:Cell Culture Techniques; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D057809:HEK293 Cells; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D011494:Protein Kinases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D013997:Time Factors
D055767:Immobilized Proteins; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases