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 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 »
- Authors:
-
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Fundamental and Computational Sciences Directorate
- Publication Date:
- Research Org.:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1167286
- Alternate Identifier(s):
- OSTI ID: 1233933
- Report Number(s):
- PNNL-SA-104877
Journal ID: ISSN 1046-5928; 47735; 44691; 41891; 48235; 19851a; 400412000
- Grant/Contract Number:
- AC05-76RL01830; DE-015347
- Resource 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
- 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
Citation Formats
Buchko, Garry W., and Shaw, Wendy J. Improved protocol to purify untagged amelogenin – Application to murine amelogenin containing the equivalent P70→T point mutation observed in human amelogenesis imperfecta. United States: N. p., 2014.
Web. doi:10.1016/j.pep.2014.09.020.
Buchko, Garry W., & Shaw, Wendy J. Improved protocol to purify untagged amelogenin – Application to murine amelogenin containing the equivalent P70→T point mutation observed in human amelogenesis imperfecta. United States. https://doi.org/10.1016/j.pep.2014.09.020
Buchko, Garry W., and Shaw, Wendy J. Mon .
"Improved protocol to purify untagged amelogenin – Application to murine amelogenin containing the equivalent P70→T point mutation observed in human amelogenesis imperfecta". United States. https://doi.org/10.1016/j.pep.2014.09.020. https://www.osti.gov/servlets/purl/1167286.
@article{osti_1167286,
title = {Improved protocol to purify untagged amelogenin – Application to murine amelogenin containing the equivalent P70→T point mutation observed in human amelogenesis imperfecta},
author = {Buchko, Garry W. and Shaw, Wendy J.},
abstractNote = {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 P71T 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.},
doi = {10.1016/j.pep.2014.09.020},
journal = {Protein Expression and Purification},
number = C,
volume = 105,
place = {United States},
year = {Mon Oct 13 00:00:00 EDT 2014},
month = {Mon Oct 13 00:00:00 EDT 2014}
}
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Works referencing / citing this record:
The energetic basis for hydroxyapatite mineralization by amelogenin variants provides insights into the origin of amelogenesis imperfecta
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