Enamel matrix proteins are central to the process of enamel formation, but they are not part of the product. Enamel matrix proteinases process enamel proteins into polypeptides during the secretory stage, and completely degrade the matrix during maturation. It is not possible to isolate any proteins from erupted teeth, or significant quantities of intact enamel proteins from developing teeth. The inability to obtain intact enamel proteins from length recombinant enamel proteins and specific antibodies to test hypotheses concerning the roles of proteins in dental enamel formation, the following three Specific Aims are proposed: 1) To express pig amelogenin, sheathlin, enamelin, enamelysin, and EMSP1 in bacteria and to isolate and characterize the recombinant proteins.
This aim will be accomplished using the pET11 and pPROEX1 expression systems. The proteins will be purified by successive ammonium sulfate precipitations, followed by high pressure, liquid chromatography (HPLC) using reversed phase and anion or cation exchange columns. The recombinant proteins will be characterized by amino-terminal sequencing, amino acid analysis, and laser desorption mass spectrometry. 2)To generate antibodies against recombinant amelogenin, sheathlin, enamelin, enamelysin, and EMSP1.
This aim will be accomplished using recombinant enamel proteins expressed in bacteria. The purified antigens will be infected into rabbits and characterize by ELISA. The rabbit serum will be absorbed by bacterial extracts to remove antibodies reactive against E. coli proteins and then purified on an FPLC affinity column to isolate only the IgG fraction. 3) To express pig amelogenin, sheathlin, enamelin, enamelysin, and EMSP1 in eukaryotic systems and to isolate and characterize the expression products This aim will be accomplished using the pcDNA3.1/Zeo, pCl-Neo, and nADLOX.HTM expression systems. All of these systems are used to express proteins in mammalian cells, to most accurately duplicate the post-translational modifications that occur to enamel proteins in vivo. The proteins will be expressed with signal peptides for secretion by the host cell, and purified by and preparative FPLC immunoaffinity chromatography. The expressed proteins will be characterized to determine the nature of their post-translational modifications.
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