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Purification of A-Raf and structural studies of mannitol dehydrogenase



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The work herein describes the research of two separate projects: the purification of A-Raf and the crystallization and X-ray diffraction of Thermotoga maritima mannitol dehydrogenase (TmMtDH). A-Raf is a one of three Raf isoforms of serine/threonine kinases involved in the mitogen-activated protein kinase (MAPK) pathway, a cell proliferation pathway that has been associated with many cancers. In addition, only the A-Raf isoform can uniquely bind to the regulatory subunit of phosphatidylinositol-3-kinase (PI3K), which is part of the Akt/PI3K pathway and is another important signaling molecule deregulated in human cancers. Therefore, the main focus of this study was to purify and crystallize this protein in order to characterize what makes A-Raf structurally unique from the other two Raf isoforms. Several portions of A-Raf were purified throughout this study, but most research concentrated on the conserved region 2 and 3 (CR2 and CR3) domains of A-Raf and the full-length protein. The CR2/CR3 domains and full-length A-Raf were purified by affinity chromatography on a glutathione Sepharose column and column fractions were analyzed by SDS-PAGE. Two different bands measuring approximately 75 kDa and 66 kDa resolved on the SDS-PAGE gel of full-length A-Raf while three bands measuring approximately 75 kDa, 66 kDa and 45 kDa resolved on the SDS-PAGE gel of the CR2/CR3 domains of A-Raf. The CR2/CR3 domains and full-length A-Raf were also extensively studied by mass spectrometry but results were inconclusive. Western blot analysis was also performed on the CR2/CR3 domains and full-length A-Raf. Results indicated that multiple bands were present and that degradation of the protein had taken place. A-Raf was thus deemed unsuitable for crystallization trials and the project was terminated. Mannitol is an acyclic polyalcohol and is used commercially for several purposes including acting as an osmoregulatory compound in several pharmaceuticals and as an artificial sweetener in products targeted for diabetic patients. Commercially, mannitol is produced by the hydrogenation of 50% fructose/50% glucose syrup at high temperatures. However, the product of this process yields an excess of sorbitol and therefore the mannitol requires further purification. Mannitol dehydrogenase catalyzes the conversion of D-fructose to D-mannitol and has therefore been targeted for studies to produce a commercial mannitol bioreactor. The aims of this study included crystallization of the hyperthermophilic Thermotoga maritima (TmMtDH) mannitol dehydrogenase and subsequent X-ray diffraction and structure analysis. Dr. Claire Vieille at Michigan State University provided purified protein for crystallization trials. Two conditions produced diffraction quality crystals of TmMtDH. Condition 1 crystals grew in a solution containing 30% 2-methyl-2,4-pentanediol (MPD) plus 0.1 M HEPES-Na at pH 7.5. Condition 2 crystals grew in a solution containing 15-20% (w/v) polyethylene glycol (PEG) 4000 or 8000 plus 0.1 M sodium citrate at pH 4, 0.2 M sodium bromide and 10% glycerol. Crystals were flash cooled in liquid nitrogen and diffracted on the in-house diffractometer at the Saskatchewan Structural Sciences Center and at beamline 08ID-1 at the Canadian Light Source. Data were collected to 3.3 Å for the crystal that grew in condition 1 but the structure could not be solved before the completion of this project. The space group of the condition 1 crystal was P212121 with unit cell dimensions a = 83.43 Å, b = 120.61 Å, c = 145.76 Å.



Mannitol Dehydrogenase, A-Raf



Master of Science (M.Sc.)






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