title: Improved protocol to purify untagged amelogenin - Application to murine amelogenin containing the equivalent P70→T point mutation observed in human amelogenesis imperfecta.
abstract: Amelogenin is the predominant extracellular protein responsible for converting carbonated hydroxyapatite into dental enamel, the hardest and most heavily mineralized tissue in vertebrates. Despite much effort, the precise mechanism by which amelogenin regulates enamel formation is not fully understood. To assist efforts aimed at understanding the biochemical mechanism of enamel formation, more facile protocols to purify recombinantly expressed amelogenin, ideally without any tag to assist affinity purification, are advantageous. Here we describe an improved method to purify milligram quantities of amelogenin that exploits its high solubility in 2% glacial acetic acid under conditions of low ionic strength. The method involves heating the frozen cell pellet for two 15min periods at ∼70°C with 2min of sonication in between, dialysis twice in 2% acetic acid (1:250 v/v), and reverse phase chromatography. A further improvement in yield is obtained by resuspending the frozen cell pellet in 6M guanidine hydrochloride in the first step. The acetic acid heating method is illustrated with a murine amelogenin containing the corresponding P70→T point mutation observed in an human amelogenin associated with amelogenesis imperfecta (P71T), while the guanidine hydrochloride heating method is illustrated with wild type murine amelogenin (M180). The self-assembly properties of P71T were probed by NMR chemical shift perturbation studies as a function of protein (0.1-1.8mM) and NaCl (0-367mM) concentration. Relative to similar studies with wild type murine amelogenin, P71T self-associates at lower protein or salt concentrations with the interactions initiated near the N-terminus.
authors.affiliation: Fundamental and Computational Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA 99352, USA. Electronic address: garry.buchko@pnnl.gov.
authors.forename: Garry W
authors.initials: GW
authors.lastname: Buchko
authors.affiliation: Fundamental and Computational Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
authors.forename: Wendy J
authors.initials: WJ
authors.lastname: Shaw
mesh_terms: D000567:Amelogenesis Imperfecta / Q000235:genetics*; D053523:Amelogenin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002113:Calcification, Physiologic / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics*; D012965:Sodium Chloride; D013552:Swine
chemical_list: D053523:Amelogenin; D012965:Sodium Chloride
keywords: Amelogenesis imperfecta; Amelogenin; Biomineralization; Intrinsic disorder; NMR spectroscopy; Tooth enamel