Purification and in vitro characterization of zinc finger recombinases

Olorunniji, F. J., Rosser, S. J. and Stark, W. M. (2017) Purification and in vitro characterization of zinc finger recombinases. In: Eroshenko, N. (ed.) Site-Specific Recombinases: Methods and Protocols. Series: Methods in molecular biology (1642). Humana Press, pp. 229-245. ISBN 9781493971671 (doi: 10.1007/978-1-4939-7169-5_15)

Abstract

Zinc finger recombinases (ZFRs) are designer site-specific recombinases that have been adapted for a variety of genome editing purposes. Due to their modular nature, ZFRs can be customized for targeted sequence recognition and recombination. There has been substantial research on the in vivo properties and applications of ZFRs; however, in order to fully understand and customize them, it will be necessary to study their properties in vitro. Experiments in vitro can allow us to optimize catalytic activities, improve target specificity, measure and minimize off-target activity, and characterize key steps in the recombination pathway that might be modified to improve performance. Here, we present a straightforward set of protocols for the expression and purification of ZFRs, an assay system for catalytic proficiency in vitro and bandshift assays for detection of sequence-specific DNA interactions.

• Item Type: Book Sections
• Status: Published
• Glasgow Author(s) Enlighten ID: Stark, Professor Marshall and Olorunniji, Dr Femi and Rosser, Professor Susan
• Authors: Olorunniji, F. J., Rosser, S. J., and Stark, W. M.
• College/School: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Publisher: Humana Press
• ISBN: 9781493971671