Production of Recombinant Tuftelin protein for Structural and Functional Characterization

Objectives: production of functional rHTuft+. Methods: We have produced, several recombinant tuftelin proteins in eukaryotic insect Sf9 cells, using the GATEWAY and the baculovirus expression systems. Two bovine recombinant tuftelin proteins and a recombinant human tuftelin protein (rHTuft+) were produced. Atomic force microscopy analyses of rHTuft+ suggested that tuftelin self-assemble into nonosphere-like structures, in time and temperature dependent manners. This might have a crucial role in the initiation of enamel mineralization. Indirect Immunohistochemistry of mouse embryonic craniofacial complex revealed that tuftelin is also expressed early in embryogenesis in several mineralizing and non-mineralizing tissues and developing organs. Results: Preliminary studies on the role of rHTuft+ in epithelial-mesenchymal interactions were obtained using the ex-vivo mouse embryonic mandibular explant culture. rHTuft+ coated beads were introduced onto different developing mandible regions. In the developing tooth bud region, epithelial cells seemed to emerge from the oral epithelium and surround the tuftelin beads exhibiting specific morphological change. The same morphologic phenomenon was observed with epithelial cells of the salivary gland duct. Mandibular mesenchymal cells seemed to surround the rHTuft+-coated beads and to have a specific polarizing orientation. No such reactions were observed when BSA-coated beads were used. Conclusions: for the first time, we produced full length recombinant tuftelin proteins. rHtuft+ retained its functionality and effect on epithelial-mesenchymal interactions in mouse embryonic mandibular explant culture. Tuftelin expression in several odontogenic and non-odontogenic mineralizing and non-mineralizing embryonic tissues was also demonstrated. These findings support the hypothesis that tuftelin is multi-functional, and might also play an important role during organogenesis, perhaps by means of epithelial-mesenchymal interactions. This Work was supported by E.U.-Framework5 "MATRIX" grant no:QLK3-CT-2001-00090.