ISOLATION AND CHARACTERIZATION OF A MOUSE AMELOGENIN EXPRESSED IN ESCHERICHIA-COLI

A mouse cDNA encoding a 180 amino acid amelogenin was subcloned into t he pET expression plasmid (Novagen, Madison, WI) for production in Esc herichia coli. A simple growth and purification protocol yields 20-50 mg of 95-99% pure recombinant amelogenin from a 4.5-liter culture. Thi s is the first heterologous expression of an enamel protein. The expre ssed protein was characterized by partial Edman sequencing, amino acid composition analysis, SDS-PAGE, Western blotting, laser desorption ma ss spectrometry, and hydroxyapatite binding. The recombinant amelogeni n is 179 amino acids in length, has a molecular weight of 20,162 dalto ns, and hydroxyapatite binding properties similar to the porcine 173 r esidue amelogenin. Solubility analyses showed that the bacterially exp ressed protein is only sparingly soluble in the pH range of 6.4-8.0 or in solutions 20% saturated with ammonium sulfate. The purified protei n was used to generate rabbit polyclonal anti-amelogenin antibodies wh ich show specific reaction to amelogenins in both Western blot analyse s of enamel extracts and in immunostaining of developing mouse molars.