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Isolation and characterization of a mouse amelogenin expressed in Escherichia coli

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Abstract

A mouse cDNA encoding a 180 amino acid amelogenin was subcloned into the pET expression plasmid (Novagen, Madison, WI) for production in Escherichia coli. A simple growth and purification protocol yields 20–50 mg of 95–99% pure recombinant amelogenin from a 4.5-liter culture. This is the first heterologous expression of an enamel protein. The expressed protein was characterized by partial Edman sequencing, amino acid composition analysis, SDS-PAGE, Western blotting, laser desorption mass spectrometry, and hydroxyapatite binding. The recombinant amelogenin is 179 amino acids in length, has a molecular weight of 20,162 daltons, and hydroxyapatite binding properties similar to the porcine 173 residue amelogenin. Solubility analyses showed that the bacterially expressed protein is only sparingly soluble in the pH range of 6.4–8.0 or in solutions 20% saturated with ammonium sulfate. The purified protein was used to generate rabbit polyclonal anti-amelogenin antibodies which show specific reaction to amelogenins in both Western blot analyses of enamel extracts and in immunostaining of developing mouse molars.

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References

  1. Termine JD, Belcourt AB, Christner PJ, Conn KM, Nylen MU (1980) Properties of dissociatively extracted fetal tooth matrix proteins. I. Principal molecular species in developing bovine enamel. J Biol Chem 255:9760–9768

    Google Scholar 

  2. Herold RC, Rosenbloom J, Granovsky M (1989) Phylogenetic distribution of enamel proteins: immunohistochemical localization with monoclonal antibodies indicates the evolutionary appearance of enamelins prior to amelogenins. Calcif Tissue Int 45:88–94

    Google Scholar 

  3. Niklas-Dahl LM, Nakahori Y, Nakagome Y, Backman B, Landegren U, Pettersson U (1991) A deletion in the amelogenin gene (AMG) causes X-linked amelogenesis imperfecta (AIH1). Genomics 10:971–975

    Google Scholar 

  4. Aldred MJ, Crawford PM, Roberts E, Thomas NST (1992) Identification of a nonsense mutation in the amelogenin gene (AMELX) in a family with X-linked amelogenesis imperfecta (AIH1). Hum Genet 90:413–416

    Google Scholar 

  5. Diekwisch T, Sasson D, Bringas Jr P, Santos V, Slavkin HC (1993) Antisense inhibition of AMEL translation demonstrates supramolecular controls for enamel HAP crystal growth during embryonic mouse molar development. Development 117:471–482

    Google Scholar 

  6. Fincham AG, Lau EC, Simmer JP, Zeichner-David M (1992) Amelogenin biochemistry—form and function. In: Slavkin HC, Price P (eds) Chemistry and biology of mineralized tissues. Elsevier Scientific Publishers, Amsterdam, Netherlands, pp 187–200

    Google Scholar 

  7. Lau EC, Simmer JP, Bringas P Jr, Hsu D, Hu C-C, Zeichner-David M, Thiemann F, Snead ML, Slavkin HC, Fincham AG (1992) Alternative splicing of the mouse amelogenin primary RNA transcript contributes to amelogenin heterogeneity. Biochem Biophys Res Commun 188(3):1253–1260

    Google Scholar 

  8. Nikiforuk G, Simmons NS (1965) Purification and properties of protein from embronic bovine enamel. J Dent Res 44:1119–1122

    Google Scholar 

  9. Sasaki S, Takagi T, Suzuki M (1991) Cyclical changes in pH in bovine developing enamel as sequential bands. Archs Oral Biol 36:227–231

    Google Scholar 

  10. Shimokawa H, Sobel ME, Sasaki M, Termine JD, Young MF (1987) Heterogeneity of amelogenin mRNA in the bovine tooth germ. J Biol Chem 262:4042–4047

    Google Scholar 

  11. Young MF, Shimokawa HS, Sobel ME, Termine JD (1987) A characterization of amelogenin messenger RNA in the bovine tooth germ. Adv Dent Res 1:289–292

    Google Scholar 

  12. Gibson CW, Golub E, Ding W, Shimokawa H, Young M, Termine J, Rosenbloom J (1991) Identification of the leucine-rich amelogenin peptide (LRAP) as the translation product of an alternatively spliced transcript. Biochem Biophys Res Commun 174:1306–1312

    Google Scholar 

  13. Yamakoshi Y, Tanabe T, Fukae M, Shimizu M (1989) Amino acid sequence of porcine 25kDa amelogenin. In: Fearnhead RW (ed) Tooth Enamel. Florence Publishers, Yokahama, Japan, pp 314–318

    Google Scholar 

  14. Lau EC, Thuluvancheri K, Mohandas T, Shapiro LJ, Slavkin HC, Snead ML (1989) Human and mouse amelogenin gene loci are on the sex chromosomes. Genomics 4:162–168

    Google Scholar 

  15. Chapman VM, Keitz BT, Disteche CM, Lau EC, Snead ML (1991) Linkage of amelogenin (Amel) to the distal portion of the mouse X chromosome. Genomics 10:23–28

    Google Scholar 

  16. Salido EC, Yen PH, Koprivnikar K, Yu LC, Shapiro LJ (1991) The human enamel protein gene amelogenin is expressed from both the X and the Y chromosomes. Am J Hum Genet 50:303–316

    Google Scholar 

  17. Nakahori Y, Takenaka O, Takagome Y (1991) A human X-Y homologous region encodes “amelogenin”. Genomics 9:264–269

    Google Scholar 

  18. Fincham AG, Bessem CC, Lau EC, Pavlova Z, Shuler C, Slavkin HC, Snead ML (1991) Human developing enamel proteins exhibit a sex-linked dimorphism. Calcif Tissue Int 48:288–290

    Google Scholar 

  19. Fukae M, Tanabe T, Ijiri H, Shimizu M (1980) Studies on procine enamel proteins: a possible original enamel protein. Tsurumi Univ Dent J 6:87–94

    Google Scholar 

  20. Tanabe T, Fukae M, Uchida T, Simizu M (1992) The localization and characterization of proteinases for the initial cleavage of porcine amelogenin. Calcif Tissue Int 51:213–217

    Google Scholar 

  21. Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. In: Goeddel DV (ed) Methods in enzymology 185. Academic Press Inc, San Diego, pp 60–89

    Google Scholar 

  22. Fincham AG, Hu Y, Lau EC, Slavkin HC, Snead ML (1991) Amelogenin post-secretory processing during biomineralization in the postnatal mouse molar tooth. Archs Oral Biol 36:305–317

    Google Scholar 

  23. Keough T, Takigiku R, Lacey MP, Purdon MP (1992) Matrix-assisted laser desorption mass spectrometry of proteins isolated by capillary zone electrophoresis. Anal Chem 64:1594–1600

    Google Scholar 

  24. Beavis RC, Chait BT (1990) Rapid, sensitive analysis of protein mixtures by mass spectrometry. Proc Natl Acad Sci USA 87: 6873–6877

    Google Scholar 

  25. Beavis RC, Chait BT (1990) High-accuracy molecular mass determination of proteins using matrix-assisted laser desorption mass spectrometry. Anal Chem 62:1836–1840

    Google Scholar 

  26. Aoba T, Fukae M, Tanabe T, Shimizu M, Moreno EC (1987) Selective adsorption of porcine-amelogenins onto hydroxyapatite and their inhibitory activity on hydroxyapatite growth in supersaturated solutions. Calcif Tissue Int 41:281–289

    Google Scholar 

  27. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275

    Google Scholar 

  28. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p B 21

    Google Scholar 

  29. Scopes RK (1987) Protein purification principles and practice. Springer-Verlag, New York, p 303

    Google Scholar 

  30. Tsunasaw S, Stewart JW, Sherman FS (1985) Amino-terminal processing of mutant forms of yeast iso-1-cytochrome c: the specificities of methionine aminopeptidase and acetyltransferase. J Biol Chem 260:5382–5391

    Google Scholar 

  31. Miller CG (1987) Protein degradation and proteolytic modification. In: Neidhardt FC (ed) Escherichia coli and Salmonella typhimurium cellular and molecular biology. American Society for Microbiology, p 687

  32. Lee TD, Vemuri S, (1991) Proc of the 39th ASMS Conf on Mass Spectrometry and Allied Topics, Nashville, TN, May 19–24

  33. Slavkin HC, Zeichner-David M, MacDougall M, Bringas P, Bessem C, Honig L (1982) Antibodies to murine amelogenins: localization of enamel proteins during tooth organ development in vitro. Differentiation 23:73–82

    Google Scholar 

  34. Inai T, Kukita T, Ohsaki Y, Kengo N, Kukita A, Kurisu K (1991) Immunohistochemical demonstration of amelogenin penetration toward the dental pulp in the early stages of ameloblast development in rat molar tooth germs. Anat Rec 229:259–270

    Google Scholar 

  35. Nakamura M, Bringas P, Nanci A, Zeichner-David M, Ashdown B, Slavkin HC (in press) Translocation of enamel proteins from inner enamel epithelia to odontoblasts during mouse tool development. Anat Rec

  36. Aoba T, Tanabe T, Moreno EC (1987) Function of amelogenins in porcine enamel mineralization during the secretory stage of amelogenesis. Adv Dent Res 1(2):252–260

    Google Scholar 

  37. Samachson J (1969) Basic requirements for calcification. Nature 221:147–1248

    Google Scholar 

  38. Eastoe J (1989) Tooth enamel V. In: Fearnhead RW (ed) Florence Publishers, Yokahama, Japan, p 89

    Google Scholar 

  39. Watson M (1960) The extracellular nature of enamel in the rat. J Biophys Biochem Cytol 7:489–492

    Google Scholar 

  40. Dogterom AA, Bonckers ALJJ (1983) Carbonic anhydrase in developing hamster molars. J Dent Res 62:789–791

    Google Scholar 

  41. Snead ML, Luo W, Lau EC, Slavkin HC (1988) Spatial- and temporal-restricted pattern for amelogenin gene expression during mouse molar tooth organogenesis. Development 104:77–85

    Google Scholar 

  42. Slavkin HC, Bessem C, Bringas P, Zeichner-David M, Nanci A, Snead ML (1988) Sequential expression and differential function of multiple enamel proteins during fetal, neonatal, and early postnatal stages of mouse molar organogenesis. Differentiation 37:26–39

    Google Scholar 

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Author notes

  1. J. P. Simmer

    Present address: Health Science Center at San Antonio, Department of Pediatric Dentistry, University of Texas School of Dentistry, 7703 Floyd Curl Drive, 78284-7888, San Antonio, TX, USA

Authors and Affiliations

  1. Center for Craniofacial Molecular Biology, University of Southern California, School of Dentistry, 2250 Alcazar Street, CSA 1st Floor, 90033, Los Angeles, California, USA

    J. P. Simmer, E. C. Lau, C. C. Hu, M. Zeichner-David, M. L. Snead, H. C. Slavkin & A. G. Fincham

  2. Forsyth Dental Center, Forsyth Research Institute, 140 Fenway, 02115, Boston, Massachusetts, USA

    T. Aoba

  3. Miami Valley Laboratories, The Procter & Gamble Company, P.O. Box 398707, 45239-8707, Cincinnati, Ohio, USA

    M. Lacey & D. Nelson

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  1. J. P. Simmer
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  2. E. C. Lau
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  3. C. C. Hu
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  4. T. Aoba
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  7. M. Zeichner-David
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  8. M. L. Snead
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  9. H. C. Slavkin
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Simmer, J.P., Lau, E.C., Hu, C.C. et al. Isolation and characterization of a mouse amelogenin expressed in Escherichia coli . Calcif Tissue Int 54, 312–319 (1994). https://doi.org/10.1007/BF00295956

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  • DOI: https://doi.org/10.1007/BF00295956

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