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

Buchko, Garry W.; Shaw, Wendy J.

doi:10.1016/j.pep.2014.09.020

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

Journal Article · Mon Oct 13 00:00:00 EDT 2014 · Protein Expression and Purification

DOI:https://doi.org/10.1016/j.pep.2014.09.020· OSTI ID:1167286

Buchko, Garry W. ^[1]; Shaw, Wendy J. ^[1]

Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Fundamental and Computational Sciences Directorate

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.

View Accepted Manuscript (DOE)

Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States), Environmental Molecular Sciences Laboratory (EMSL)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)

Grant/Contract Number:: AC05-76RL01830

OSTI ID:: 1167286

Alternate ID(s):: OSTI ID: 1233933

Report Number(s):: PNNL-SA--104877; 47735; 44691; 41891; 48235; 19851a; 400412000

Journal Information:: Protein Expression and Purification, Journal Name: Protein Expression and Purification Journal Issue: C Vol. 105; ISSN 1046-5928

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

References (52)

Interaction of amelogenin with hydroxyapatite crystals: An adherence effect through amelogenin molecular self-association Moradian-Oldak, J.; Tan, J.; Fincham, A. G. Biopolymers, Vol. 46, Issue 4 https://doi.org/10.1002/(SICI)1097-0282(19981005)46:4<225::AID-BIP4>3.0.CO;2-R	journal	October 1998
Detection of monodisperse aggregates of a recombinant amelogenin by dynamic light scattering Moradian-Oldak, J.; Simmer, J. P.; Lau, E. C. Biopolymers, Vol. 34, Issue 10 https://doi.org/10.1002/bip.360341006	journal	October 1994
Modification of calcium-phosphate coatings on titanium by recombinant amelogenin Wen, H. B.; Moradian-Oldak, J. Journal of Biomedical Materials Research, Vol. 64A, Issue 3 https://doi.org/10.1002/jbm.a.10401	journal	February 2003
Probing the self-association, intermolecular contacts, and folding propensity of amelogenin: TFE-Induced Folding Propensities of Amelogenin Ndao, Moise; Dutta, Kaushik; Bromley, Keith M. Protein Science, Vol. 20, Issue 4 https://doi.org/10.1002/pro.603	journal	March 2011
Self-Assembly of a Recombinant Amelogenin Protein Generates Supramolecular Structures Fincham, A. G.; Moradian-Oldak, J.; Simmer, J. P. Journal of Structural Biology, Vol. 112, Issue 2 https://doi.org/10.1006/jsbi.1994.1011	journal	March 1994
Evidence for Amelogenin "Nanospheres" as Functional Components of Secretory-Stage Enamel Matrix Fincham, A. G.; Moradian-Oldak, J.; Diekwisch, T. G. H. Journal of Structural Biology, Vol. 115, Issue 1 https://doi.org/10.1006/jsbi.1995.1029	journal	July 1995
Temperature and pH-Dependent Supramolecular Self-Assembly of Amelogenin Molecules: A Dynamic Light-Scattering Analysis Moradian-Oldak, J.; Leung, W.; Fincham, A. G. Journal of Structural Biology, Vol. 122, Issue 3 https://doi.org/10.1006/jsbi.1998.4008	journal	January 1998
1H, 13C and 15N chemical shift referencing in biomolecular NMR Wishart, David S.; Bigam, Colin G.; Yao, Jian Journal of Biomolecular NMR, Vol. 6, Issue 2 https://doi.org/10.1007/BF00211777	journal	September 1995
Isolation and characterization of a mouse amelogenin expressed in Escherichia coli Simmer, J. P.; Lau, E. C.; Hu, C. C. Calcified Tissue International, Vol. 54, Issue 4 https://doi.org/10.1007/BF00295956	journal	April 1994
Immunochemical and immunohistochemical studies, using antisera against porcine 25 kDa amelogenin, 89 kDa enamelin and the 13?17 kDa nonamelogenins, on immature enamel of the pig and rat Uchida, T.; Tanabe, T.; Fukae, M. Histochemistry, Vol. 96, Issue 2 https://doi.org/10.1007/BF00315983	journal	June 1991
Length and shape of enamel crystals Daculsi, G.; Menanteau, J.; Kerebel, L. M. Calcified Tissue International, Vol. 36, Issue 1 https://doi.org/10.1007/BF02405364	journal	December 1984
1H, 13C, and 15N resonance assignments of murine amelogenin, an enamel biomineralization protein Buchko, Garry W.; Bekhazi, Jacky; Cort, John R. Biomolecular NMR Assignments, Vol. 2, Issue 1 https://doi.org/10.1007/s12104-008-9092-x	journal	April 2008
DNA sequence for cloned cDNA for murine amelogenin reveal the amino acid sequence for enamel-specific protein Snead, Malcolm L.; Lau, Eduardo C.; Zeichner-David, Margarita Biochemical and Biophysical Research Communications, Vol. 129, Issue 3 https://doi.org/10.1016/0006-291X(85)91964-3	journal	June 1985
A simple method for displaying the hydropathic character of a protein Kyte, Jack; Doolittle, Russell F. Journal of Molecular Biology, Vol. 157, Issue 1, p. 105-132 https://doi.org/10.1016/0022-2836(82)90515-0	journal	May 1982
Interior and surface of monomeric proteins Miller, Susan; Janin, Joël; Lesk, Arthur M. Journal of Molecular Biology, Vol. 196, Issue 3 https://doi.org/10.1016/0022-2836(87)90038-6	journal	August 1987
Autosomal recessive pigmented hypomaturation amelogenesis imperfecta Witkop, Carl J.; Kuhlmann, William; Sauk, John Oral Surgery, Oral Medicine, Oral Pathology, Vol. 36, Issue 3 https://doi.org/10.1016/0030-4220(73)90215-6	journal	September 1973
An amelogenin gene defect associated with human X-linked amelogenesis imperfecta Collier, P. M.; Sauk, J. J.; Rosenbloom, J. Archives of Oral Biology, Vol. 42, Issue 3 https://doi.org/10.1016/S0003-9969(96)00099-4	journal	March 1997
Properties of dissociatively extracted fetal tooth matrix proteins. I. Principal molecular species in developing bovine enamel. Termine, J. D.; Belcourt, A. B.; Christner, P. J. Journal of Biological Chemistry, Vol. 255, Issue 20 https://doi.org/10.1016/S0021-9258(18)43458-8	journal	October 1980
Reduced hydrolysis of amelogenin may result in X-linked amelogenesis imperfecta Li, Wu; Gibson, Carolyn W.; Abrams, William R. Matrix Biology, Vol. 19, Issue 8 https://doi.org/10.1016/S0945-053X(00)00121-9	journal	January 2001
Progressive accretion of amelogenin molecules during nanospheres assembly revealed by atomic force microscopy Wen, H. B.; Fincham, A. G.; Moradian-Oldak, J. Matrix Biology, Vol. 20, Issue 5-6 https://doi.org/10.1016/S0945-053X(01)00144-5	journal	September 2001
Regulated gene expression dictates enamel structure and tooth function Paine, Michael L.; White, Shane N.; Luo, Wen Matrix Biology, Vol. 20, Issue 5-6 https://doi.org/10.1016/S0945-053X(01)00153-6	journal	September 2001
Is there more to enamel matrix proteins than biomineralization? Zeichner-David, Margarita Matrix Biology, Vol. 20, Issue 5-6 https://doi.org/10.1016/S0945-053X(01)00155-X	journal	September 2001
Interfacial aspects of biomineralization Hunter, Graeme K. Current Opinion in Solid State and Materials Science, Vol. 1, Issue 3 https://doi.org/10.1016/S1359-0286(96)80036-2	journal	June 1996
A solution NMR investigation into the impaired self-assembly properties of two murine amelogenins containing the point mutations T21→I or P41→T Buchko, Garry W.; Lin, Genyao; Tarasevich, Barbara J. Archives of Biochemistry and Biophysics, Vol. 537, Issue 2 https://doi.org/10.1016/j.abb.2013.07.015	journal	September 2013
Altered self-assembly and apatite binding of amelogenin induced by N-terminal proline mutation Zhu, Li; Uskoković, Vuk; Le, Thuan Archives of Oral Biology, Vol. 56, Issue 4 https://doi.org/10.1016/j.archoralbio.2010.10.017	journal	April 2011
The nucleation and growth of calcium phosphate by amelogenin Tarasevich, Barbara J.; Howard, Christopher J.; Larson, Jenna L. Journal of Crystal Growth, Vol. 304, Issue 2 https://doi.org/10.1016/j.jcrysgro.2007.02.035	journal	June 2007
The effect of recombinant mouse amelogenins on the formation and organization of hydroxyapatite crystals in vitro Beniash, Elia; Simmer, James P.; Margolis, Henry C. Journal of Structural Biology, Vol. 149, Issue 2 https://doi.org/10.1016/j.jsb.2004.11.001	journal	February 2005
pH triggered self-assembly of native and recombinant amelogenins under physiological pH and temperature in vitro Wiedemann-Bidlack, Felicitas B.; Beniash, Elia; Yamakoshi, Yasuo Journal of Structural Biology, Vol. 160, Issue 1 https://doi.org/10.1016/j.jsb.2007.06.007	journal	October 2007
The leucine rich amelogenin protein (LRAP) adsorbs as monomers or dimers onto surfaces Tarasevich, Barbara J.; Lea, Scott; Shaw, Wendy J. Journal of Structural Biology, Vol. 169, Issue 3 https://doi.org/10.1016/j.jsb.2009.10.007	journal	March 2010
Protein production by auto-induction in high-density shaking cultures Studier, F. William Protein Expression and Purification, Vol. 41, Issue 1, p. 207-234 https://doi.org/10.1016/j.pep.2005.01.016	journal	May 2005
Histidine tag fusion increases expression levels of active recombinant amelogenin in Escherichia coli Svensson, Johan; Andersson, Christer; Reseland, Janne E. Protein Expression and Purification, Vol. 48, Issue 1 https://doi.org/10.1016/j.pep.2006.01.005	journal	July 2006
Phosphorylation and Ionic Strength Alter the LRAP–HAP Interface in the N-Terminus Lu, Jun-xia; Xu, Yimin Sharon; Shaw, Wendy J. Biochemistry, Vol. 52, Issue 13 https://doi.org/10.1021/bi400071a	journal	March 2013
Functional and Structural Characterization of DR_0079 from Deinococcus radiodurans , a Novel Nudix Hydrolase with a Preference for Cytosine (Deoxy)ribonucleoside 5′-Di- and Triphosphates ^† Buchko, Garry W.; Litvinova, Olga; Robinson, Howard Biochemistry, Vol. 47, Issue 25 https://doi.org/10.1021/bi800099d	journal	June 2008
The Tooth Enamel Protein, Porcine Amelogenin, Is an Intrinsically Disordered Protein with an Extended Molecular Configuration in the Monomeric Form ^† Delak, Katya; Harcup, Craig; Lakshminarayanan, Rajamani Biochemistry, Vol. 48, Issue 10 https://doi.org/10.1021/bi802175a	journal	March 2009
Neutron Reflectometry Studies of the Adsorbed Structure of the Amelogenin, LRAP Tarasevich, Barbara J.; Perez-Salas, Ursula; Masica, David L. The Journal of Physical Chemistry B, Vol. 117, Issue 11 https://doi.org/10.1021/jp311936j	journal	March 2013
Identification and characterization of amelogenin genes in monotremes, reptiles, and amphibians Toyosawa, S.; O'hUigin, C.; Figueroa, F. Proceedings of the National Academy of Sciences, Vol. 95, Issue 22 https://doi.org/10.1073/pnas.95.22.13056	journal	October 1998
Specific Amelogenin Gene Splice Products Have Signaling Effects on Cells in Culture and in Implants in Vivo Veis, Arthur; Tompkins, Kevin; Alvares, Keith Journal of Biological Chemistry, Vol. 275, Issue 52 https://doi.org/10.1074/jbc.M002308200	journal	December 2000
Perturbed Amelogenin Secondary Structure Leads to Uncontrolled Aggregation in Amelogenesis Imperfecta Mutant Proteins Lakshminarayanan, Rajamani; Bromley, Keith M.; Lei, Ya-Ping Journal of Biological Chemistry, Vol. 285, Issue 52 https://doi.org/10.1074/jbc.M110.131136	journal	October 2010
Dissecting Amelogenin Protein Nanospheres: CHARACTERIZATION OF METASTABLE OLIGOMERS Bromley, Keith M.; Kiss, Andrew S.; Lokappa, Sowmya Bekshe Journal of Biological Chemistry, Vol. 286, Issue 40 https://doi.org/10.1074/jbc.M111.250928	journal	August 2011
A mutation in the mouse Amelx tri-tyrosyl domain results in impaired secretion of amelogenin and phenocopies human X-linked amelogenesis imperfecta Barron, Martin J.; Brookes, Steven J.; Kirkham, Jennifer Human Molecular Genetics, Vol. 19, Issue 7 https://doi.org/10.1093/hmg/ddq001	journal	January 2010
Enamel matrix proteins; old molecules for new applications Lyngstadaas, Sp; Wohlfahrt, Jc; Brookes, Sj Orthodontics & Craniofacial Research, Vol. 12, Issue 3 https://doi.org/10.1111/j.1601-6343.2009.01459.x	journal	August 2009
Supramolecular Assembly of Amelogenin Nanospheres into Birefringent Microribbons Du, C. Science, Vol. 307, Issue 5714 https://doi.org/10.1126/science.1105675	journal	March 2005
Amelogenesis Imperfecta: Genotype-Phenotype Studies in 71 Families Wright, J. Timothy; Torain, Melody; Long, Kimberly Cells Tissues Organs, Vol. 194, Issue 2-4 https://doi.org/10.1159/000324339	journal	January 2011
Folding, Assembly, and Aggregation of Recombinant Murine Amelogenins with T21I and P41T Point Mutations Bromley, Keith M.; Lakshminarayanan, Rajamani; Lei, Ya-Ping Cells Tissues Organs, Vol. 194, Issue 2-4 https://doi.org/10.1159/000324342	journal	January 2011
Unique Enamel Phenotype Associated with Amelogenin Gene (AMELX) Codon 41 Point Mutation Ravassipour, D. B.; Hart, P. S.; Hart, T. C. Journal of Dental Research, Vol. 79, Issue 7 https://doi.org/10.1177/00220345000790070801	journal	July 2000
Biological Organization of Hydroxyapatite Crystallites into a Fibrous Continuum Toughens and Controls Anisotropy in Human Enamel White, S. N.; Luo, W.; Paine, M. L. Journal of Dental Research, Vol. 80, Issue 1 https://doi.org/10.1177/00220345010800010501	journal	January 2001
Mineral Association Changes the Secondary Structure and Dynamics of Murine Amelogenin Lu, J. X.; Xu, Y. S.; Buchko, G. W. Journal of Dental Research, Vol. 92, Issue 11 https://doi.org/10.1177/0022034513504929	journal	October 2013
Molecular Mechanisms of Dental Enamel Formation Simmer, J. P.; Fincham, A. G. Critical Reviews in Oral Biology & Medicine, Vol. 6, Issue 2 https://doi.org/10.1177/10454411950060020701	journal	April 1995
Protein Interactions During Assembly of the Enamel Organic Extracellular Matrix Paine, Michael L.; Snead, Malcolm L. Journal of Bone and Mineral Research, Vol. 12, Issue 2 https://doi.org/10.1359/jbmr.1997.12.2.221	journal	February 1997
Amelogenin Supramolecular Assembly in Nanospheres Defined by a Complex Helix-Coil-PPII Helix 3D-Structure Zhang, Xu; Ramirez, Benjamin E.; Liao, Xiubei PLoS ONE, Vol. 6, Issue 10 https://doi.org/10.1371/journal.pone.0024952	journal	October 2011
One-Step Purification of Recombinant Human Amelogenin and Use of Amelogenin as a Fusion Partner Svensson Bonde, Johan; Bulow, Leif PLoS ONE, Vol. 7, Issue 3 https://doi.org/10.1371/journal.pone.0033269	journal	March 2012
The flexible structure of the K24S28 region of Leucine-Rich Amelogenin Protein (LRAP) bound to apatites as a function of surface type, calcium, mutation, and ionic strength Lu, Jun-xia; Burton, Sarah D.; Xu, Yimin S. Frontiers in Physiology, Vol. 5 https://doi.org/10.3389/fphys.2014.00254	journal	July 2014

Cited By (1)

The energetic basis for hydroxyapatite mineralization by amelogenin variants provides insights into the origin of amelogenesis imperfecta Tao, Jinhui; Shin, Yongsoon; Jayasinha, Rajith Proceedings of the National Academy of Sciences, Vol. 116, Issue 28 https://doi.org/10.1073/pnas.1815654116	journal	June 2019

Similar Records

A solution NMR investigation into the impaired self-assembly properties of two murine amelogenins containing the point mutations T21→I or P41→T

Journal Article · Mon Aug 26 00:00:00 EDT 2013 · Archives of Biochemistry and Biophysics, 537(2):217-224 · OSTI ID:1091431

Amelogenin signal peptide mutation: Correlation between mutations in the amelogenin gene (AMGX) and manifestations of X-linked amelogenesis imperfecta

Journal Article · Tue Feb 28 23:00:00 EST 1995 · Genomics · OSTI ID:249977

The energetic basis for hydroxyapatite mineralization by amelogenin variants provides insights into the origin ofamelogenesis imperfecta

Journal Article · Tue Jun 25 00:00:00 EDT 2019 · Proceedings of the National Academy of Sciences of the United States of America · OSTI ID:1776573

Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
Environmental Molecular Sciences Laboratory
NMR spectroscopy
amelogenesis imperfecta
amelogenin
biomineralization
intrinsic disorder
tooth enamel

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

Citation Formats

References (52)

Cited By (1)

Similar Records

Related Subjects