HaloTag-based purification of functional human kinases from mammalian cells.

摘要: Although cultured mammalian cells are preferred for producing functional proteins with appropriate post-translational modifications, purification of recombinant is frequently hampered by low expression. We have addressed this creating a new method configured specifically cell culture that provides rapid detection and efficient purification. This approach based on HaloTag, protein fusion tag designed to bind rapidly, selectively covalently series synthetic ligands can carry variety groups, including fluorescent dyes or solid supports Since the binding HaloTag HaloLink resin essentially irreversible, it overcomes equilibrium-based limitations associated affinity tags enables capture target protein, even at expression levels. The released from specific cleavage using TEV protease fused (HaloTEV), leaving both HaloTEV permanently attached highly pure, tag-free in solution. enable labeling proteins, providing convenient way monitor expression, thus facilitate identification optimal transient transfection conditions as well selection high stable lines. capabilities been demonstrated five human kinases HEK293T cells. In addition, when purifications FLAG, 3xFLAG, His(6)Tag were performed parallel, was shown provide significantly higher yields, purity overall recovery expressed proteins.