Current Chemical Genomics and Translational Medicine

2012, 6 : 55-71
Published online 2012 October 5. DOI: 10.2174/1875397301206010055
Publisher ID: CCGTM-6-55

RESEARCH ARTICLE
 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 and Keith V Wood1
1 Promega Corporation, Madison, WI, USA
2 Promega Biosciences Incorporated, San Luis Obispo, CA, USA
3 Thermo Fisher Scientific, Life Science Research-Cellomics, Pittsburg, PA, USA
4 Cellular Dynamics International, Madison, WI, USA
5 GenScript USA Incorporated, Piscataway, NJ, USA
6 Gas Technology Institute, Des Plaines, IL, USA

* Address correspondence to this author at the Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA; Tel: 608-274-1181; Fax: 608-298-4818; E-mail: lance.encell@promega.com

ABSTRACT

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.

Keywords:

Affinity tag, dehalogenase, DhaA, directed evolution, fusion tag, HaloTag, protein capture, protein detection, protein engineering, protein/cellular imaging, protein isolation, protein labeling, protein purification.