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Title: Improved protocol to purify untagged amelogenin – Application to murine amelogenin containing the equivalent P70→T point mutation observed in human amelogenesis imperfecta

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 15 min periods at ~70 ºC with two minutes 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 6 M 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 P71Tmore » were probed by NMR chemical shift perturbation studies as a function of protein (0.1 to 1.8 mM) and NaCl (0 to 367 mM) concentration. In conclusion, 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.« less
Authors:
 [1] ;  [1]
  1. Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Fundamental and Computational Sciences Directorate
Publication Date:
OSTI Identifier:
1167286
Report Number(s):
PNNL-SA--104877
Journal ID: ISSN 1046-5928; 47735; 44691; 41891; 48235; 19851a; 400412000
Grant/Contract Number:
AC05-76RL01830; DE-015347
Type:
Accepted Manuscript
Journal Name:
Protein Expression and Purification
Additional Journal Information:
Journal Volume: 105; Journal Issue: C; Journal ID: ISSN 1046-5928
Publisher:
Elsevier
Research Org:
Pacific Northwest National Laboratory (PNNL), Richland, WA (United States), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY amelogenesis imperfecta; intrinsic disorder; amelogenin; tooth enamel; biomineralization; NMR spectroscopy; Environmental Molecular Sciences Laboratory