Cultured mammalian cells offer an environment well suited for producing properly folded and functional mammalian proteins with appropriate post translational modifications(1) (2). However, the low expression levels of recombinant proteins in cultured mammalian cells presents a challenge for their purification. As a result, attaining satisfactory yield and purity depends on selective and efficient capture of these proteins from the crude cell lysate.
Affinity tag-based chromatography is commonly used to facilitate protein purification. This technique uses specific and reversible interactions between the tagged protein of interest (i.e., fusion protein) and purification resin(3) (4). The equilibrium-based binding means that the protein is constantly being exchanged between the bound (to the resin) and unbound state. Since this equilibrium depends on the protein concentration and the binding affinity of the tag at low expression levels, binding efficiency may be reduced, leading to low recovery of the fusion protein. In addition, the loss of bound protein during the purification process can further reduce yield.
The HaloTag® platform provides a new purification approach based on covalent capture that addresses these limitations(5) (6). The highly specific and irreversible binding of the HaloTag® protein to the HaloLink™ Resin enables efficient protein capture regardless of the expression level and significantly reduces protein loss during washes of the resin, resulting in higher protein recovery. Since covalently bound fusion protein can’t be eluted by conventional methods, the protein of interest is released from the resin by specific cleavage at an optimized TEV recognition site. In addition, the absence of any endogenous homologs of HaloTag® protein in mammalian cells minimize concerns regarding cross-reactivity and makes for a highly selective purification process. These features likely all contribute to the higher purity and yield we achieved in this study with the HaloTag® protein tag compared to 3xFLAG® and His6tag systems. In addition, the ability of HaloTag® fusion protein to bind a series of customized ligands carrying different functional groups, including solid supports and fluorescent dyes, enables a broad range of applications such as cellular imaging, cell sorting, protein detection, interaction studies and purification(5) (6) (7) (8).