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)

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: Mezna, Dr Mokdad and Gray, Dr Christopher and Ismail, Dr Shehab and Drysdale, Dr Martin and Bower, Professor Justin
• Authors: Gray, C. H., Konczal, J., Mezna, M., Ismail, S., Bower, J., and Drysdale, M.
• College/School: College of Medical Veterinary and Life Sciences > School 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