A fully automated procedure for the parallel, multidimensional purification and nucleotide loading of the human GTPases KRas, Rac1 and RalB

Gray, C. H., Konczal, J., Mezna, M., Ismail, S. , Bower, J. and Drysdale, M. (2017) A fully automated procedure for the parallel, multidimensional purification and nucleotide loading of the human GTPases KRas, Rac1 and RalB. Protein Expression and Purification, 132, pp. 75-84. (doi: 10.1016/j.pep.2017.01.010) (PMID:28137655) (PMCID:PMC5415301)

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Abstract

Small GTPases regulate many key cellular processes and their role in human disease validates many proteins in this class as desirable targets for therapeutic intervention. Reliable recombinant production of GTPases, often in the active GTP loaded state, is a prerequisite for the prosecution of drug discovery efforts. The preparation of these active forms can be complex and often constricts the supply to the reagent intensive techniques used in structure base drug discovery. We have established a fully automated, multidimensional protein purification strategy for the parallel production of the catalytic G-domains of KRas, Rac1 and RalB GTPases in the active form. This method incorporates a four step chromatography purification with TEV protease-mediated affinity tag cleavage and a conditioning step that achieves the activation of the GTPase by exchanging GDP for the non-hydrolyzable GTP analogue GMPPnP. We also demonstrate that an automated method is efficient at loading of KRas with mantGDP for application in a SOS1 catalysed fluorescent nucleotide exchange assay. In comparison to more conventional manual workflows the automated method offers marked advantages in method run time and operator workload. This reduces the bottleneck in protein production while generating products that are highly purified and effectively loaded with nucleotide analogues.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Drysdale, Dr Martin and Bower, Dr Justin and Mezna, Dr Mokdad and Gray, Dr Christopher and Ismail, Dr Shehab
Authors: Gray, C. H., Konczal, J., Mezna, M., Ismail, S., Bower, J., and Drysdale, M.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
Journal Name:Protein Expression and Purification
Publisher:Elsevier
ISSN:1046-5928
ISSN (Online):1096-0279
Published Online:28 January 2017
Copyright Holders:Copyright © 2017 The Authors
First Published:First published in Protein Expression and Purification 132:75-84
Publisher Policy:Reproduced under a Creative Commons License

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