Production of a Secreted Recombinant Human Ameloblastin Protein

Previously, we have reported the production of the recombinant human ameloblastin protein using the baculovirus system. The protein was expressed in the cytoplasm of Spodoptera frugiperda (SF9) insect cells (Rosenfeld 2004 IADR abstract). This recombinant protein was purified and characterized by SDS PAGE, Western blot and ESI/MS/MS sequencing, confirming the production of the full length human ameloblastin protein. The yield was 0.75mg/liter of purified protein. Objective: In an attempt to improve both the yield of the recombinant protein and to mimic the correct processing of the normally secreted ameloblastin protein, we decided to produce a secreted recombinant human ameloblastin protein. Methods: A new recombinant baculovirus was generated using: (a) the human ameloblastin cDNA without the sequence coding the signal peptide, (b) the cDNA of honeybee signal peptide (causes secretion of recombinant proteins in the baculovirus system) and (c) a modified pFASTBAC1 donor vector which contains a C-terminal histidine tag. The recombinant construct was cloned using PCR and ligation methodologies. The correct structure of the recombinant vector was confirmed by sequencing, and recombinant bacmid (baculovirus genome) was generated in DH10Bac cells by transposition. Viral particles were produced by transfection of SF9 cells with the recombinant bacmid. Plaque purification and virus amplification were carried out in SF9 cells. The recombinant protein was characterized by SDS PAGE and Western blot analysis. Results: and Conclusion: A new recombinant human ameloblastin baculovirus was generated and was shown to express a secreted recombinant human ameloblastin protein. The honeybee signal peptide is generally cleaved off during secretion releasing the mature ameloblastin protein with a C-Terminal HisTag, enabling easy purification of the protein from the medium. High expression will be carried out in Trichopulsiani (High5) insect cells and the recombinant ameloblastin protein produced in the current work will be used for in-vitro and in-vivo studies. EU-Framework5 "Matrix" Grant-QLK3-CT-2001-00090.