Dataset Information

Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands.

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.

SUBMITTER: Encell LP

PROVIDER: S-EPMC3520037 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Publications

Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands.

Encell Lance P LP Friedman Ohana Rachel R Zimmerman Kris K Otto Paul P Vidugiris Gediminas G Wood Monika G MG Los Georgyi V GV McDougall Mark G MG Zimprich Chad C Karassina Natasha N Learish Randall D RD Hurst Robin R Hartnett James J Wheeler Sarah S Stecha Pete P English Jami J Zhao Kate K Mendez Jacqui J Benink Hélène A HA Murphy Nancy N Daniels Danette L DL Slater Michael R MR Urh Marjeta M Darzins Aldis A Klaubert Dieter H DH Bulleit Robert F RF Wood Keith V KV

Current chemical genomics 20121005

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 ...[more]

PMID: 23248739

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Project description:Strategies to integrate instructive biomolecular signals into a biomaterial are becoming increasingly complex and bioinspired. While a large majority of reports still use repeated treatments with soluble factors, this approach can be prohibitively costly and difficult to translate in vivo for applications where spatial control over signal presentation is necessary. Recent efforts have explored the use of covalent immobilization of biomolecules to the biomaterial, via both bulk (ubiquitous) as well as spatially-selective light-based crosslinking, as a means to both enhance stability and bioactivity. However, little is known about how processing conditions during immobilization impact the degree of unintended non-covalent interactions, or fouling, that takes place between the biomaterial and the biomolecule of interest. Here we demonstrate the impact of processing conditions for bulk carbodiimide (EDC) and photolithography-based benzophenone (BP) crosslinking on specific attachment vs. fouling of a model protein (Concanavalin A, ConA) within collagen-glycosaminoglycan (CG) scaffolds. Collagen source significantly impacts the selectivity of biomolecule immobilization. EDC crosslinking intensity and ligand concentration significantly impacted selective immobilization. For benzophenone photoimmobilization we observed that increased UV exposure time leads to increased ConA immobilization. Immobilization efficiency for both EDC and BP strategies was maximal at physiological pH. Increasing ligand concentration during immobilization process led to enhanced immobilization for EDC chemistry, no impact on BP immobilization, but significant increases in non-specific fouling. Given recent efforts to covalently immobilize biomolecules to a biomaterial surface to enhance bioactivity, improved understanding of the impact of crosslinking conditions on selective attachment versus non-specific fouling will inform the design of instructive biomaterials for applications across tissue engineering.

Dataset Information

Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands.

Publications

Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands.

Similar Datasets

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