title: A fully automated procedure for the parallel, multidimensional purification and nucleotide loading of the human GTPases KRas, Rac1 and RalB.
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.
authors.affiliation: Drug Discovery Program, CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK. Electronic address: c.gray@beatson.gla.ac.uk.
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Gray
authors.affiliation: Drug Discovery Program, CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Konczal
authors.affiliation: Drug Discovery Program, CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK.
authors.forename: Mokdad
authors.initials: M
authors.lastname: Mezna
authors.affiliation: CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK.
authors.forename: Shehab
authors.initials: S
authors.lastname: Ismail
authors.affiliation: Drug Discovery Program, CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK.
authors.forename: Justin
authors.initials: J
authors.lastname: Bower
authors.affiliation: Drug Discovery Program, CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK.
authors.forename: Martin
authors.initials: M
authors.lastname: Drysdale
mesh_terms: D006801:Humans; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020830:rac1 GTP-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D020725:ral GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
chemical_list: C117307:KRAS protein, human; C107953:RAC1 protein, human; C483163:Ralb protein, human; D011994:Recombinant Proteins; D016283:Proto-Oncogene Proteins p21(ras); D020830:rac1 GTP-Binding Protein; D020725:ral GTP-Binding Proteins
keywords: Automated protein purification; FPLC; Histidine tag; Human small GTPases; Multidimensional purification; Nucleotide exchange; Parallel purification; Protein conditioning