One-shot method for purification of multiple natural amelogenin isoforms

Amelogenin isoforms constitute the predominant component in the enamel matrix and each amelogenin isoform executes unique role in the enamel biomineralization process. Enamel matrix derivative enriching amelogenin isoforms have also bioactive property for tissue regeneration. Despite the development of recombinant protein technology that has greatly forwarded the understanding of amelogenin properties, substantial evidences have revealed biochemical and functional difference between natural amelogenins and their recombinant form. To facilitate the study of enamel formation mechanism, more facile methodology to purify multiple natural amelogenin isoforms is pursued. Here we developed an effective one-shot method via reverse phase high-performance liquid chromatography (RP-HPLC) to purify various amelogenin isoforms from pig-derived amelogenin complex. A thorough process of chromatographic condition establishment including sample analysis on analytical scale and chromatographic condition design on preparative scale was described. Three representative amelogenin isoforms (TRAP, P148, P173) were isolated in one step and their purity was confirmed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and high resolution mass spectrometry.