Our understanding of protein function has been significantly advanced through the use of genetic fusion tags which can impart unique properties to a protein of interest (e.g. GFP as a reporter, or GST as an affinity/purification tag). Although these technologies have been broadly enabling, there are often limitations for the utility of any one tag. To address this, we used rational design and molecular evolution to engineer a modular protein fusion tag, HaloTag7. The tag can form a specific and covalent bond with chloroalkane ligands, providing the means to label proteins using fluorescent chloroalkanes, or immobilize proteins by capture to chloroalkane-containing solid supports. The tag was engineered from a modified haloalkane dehalogenase (DhaA) where the covalent intermediate formed during catalysis between enzyme and haloalkane substrate becomes trapped. The reaction is highly specific for simple chloroalkane ligands, and the resulting attachment is very stable and does not suffer from the equilibrium limitations associated with affinity tags. Molecular evolution was used to optimize the tag's binding kinetics as well as its structural compatibility when fused to a protein of interest. The result is a multifunctional tag (HaloTag7) that provides high levels of soluble expression from different hosts, and enables one with the ability to efficiently label proteins in cells or rapidly capture and purify a protein of interest from complex biological environments.

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