Isolation and characterization of a mouse amelogenin expressed in Escherichia coli.
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.
Simmer
J P
JP
Center for Craniofacial Molecular Biology, University of Southern California, School of Dentistry, Los Angeles 90033.
Lau
E C
EC
Hu
C C
CC
Aoba
T
T
Lacey
M
M
Nelson
D
D
Zeichner-David
M
M
Snead
M L
ML
Slavkin
H C
HC
Fincham
A G
AG
D053523:Amelogenin; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D003746:Dental Enamel Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D017886:Durapatite / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D012995:Solubility; D014083:Tooth Germ / Q000737:chemistry
D053523:Amelogenin; C507200:Amelx protein, mouse; D000596:Amino Acids; D018076:DNA, Complementary; D003746:Dental Enamel Proteins; D011994:Recombinant Proteins; D017886:Durapatite