Cloning and Characterization of Pig DSP and DSPP cDNA and Expression of Recombinant Protein

Dentin sialophosphoprotein (DSPP) is a chimera comprised of dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Mutations in the human DSPP gene have been associated with the dentinogenesis imperfecta. Objectives: Our objectives were to a) clone DSPP cDNA from porcine developing tooth tissue, b) express the recombinant protein, purify it, and raise polyclonal antibodies against it, and c) identify DSPP in extracts of developing teeth by Western blot analyses. Methods: We constructed a unidirectional cDNA library from the pulp organ of developing pig teeth, and isolated and characterized two distinct DSPP cDNA, as well as a novel cDNA that carries only the DSP coding region. Identical sequences proved the transcripts were expressed from a single gene. Results: The first DSPP cDNA is 2525 bp, with an open reading frame of 601 codons. The second DSPP cDNA is slightly shorter, and has a different 3’ end than the first DSPP cDNA. The DSP-only transcript is 1372 bp and is generated through the use of an alternative polyadenylation signal within intron 4 that is positioned immediately following the DSP coding region. Use of this polyadenylation signal deletes the DPP coding region and places a TGA translation termination signal as the fourth codon following the exon 3-encoded segment. The open reading frame is 386 codons. The human DSPP gene shows an identical structure and is likely to express a DSP-only transcript. Conclusions: Northern blot analyses confirmed both DSPP and DSP transcripts are expressed in porcine developing teeth. DSP, DPP, and DSPP were all successfully expressed as GST-fusion proteins and antibodies were raised against recombinant pig DSP, and then used to identify DSP in dentin extracts by Western blot analyses. This study was funded by NIDCR grant DE13237.