Development of a Dehalogenase-Based Protein Fusion Tag Capable of Rapid, Selective and Covalent Attachment to Customizable Ligands
Lance P Encell*, #, 1, Rachel Friedman Ohana#, 1, Kris Zimmerman1, Paul Otto1, Gediminas Vidugiris1, Monika G Wood1, Georgyi V Los1, 3, Mark G McDougall2, Chad Zimprich1, Natasha Karassina1, Randall D Learish1, 4, Robin Hurst1, James Hartnett1, Sarah Wheeler1, Pete Stecha1, Jami English1, Kate Zhao1, 5, Jacqui Mendez1, Hélène A Benink1, Nancy Murphy1, Danette L Daniels1, Michael R Slater1, Marjeta Urh1, Aldis Darzins1, 6, Dieter H Klaubert2, Robert F Bulleit1, Keith V Wood1
Identifiers and Pagination:Year: 2012
Issue: Suppl 1
First Page: 55
Last Page: 71
Publisher Id: CCGTM-6-55
Article History:Received Date: 02/3/2012
Revision Received Date: 04/4/2012
Acceptance Date: 16/4/2012
Electronic publication date: 5/10/2012
Collection year: 2012
open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.
Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins.