Prokaryotic expression of bone sialoprotein and identification of casein kinase II phosphorylation sites

Bone sialoprotein is an extracellular noncollagenous acidic protein that plays a role in bone mineralization and remodeling. Its expression is restricted to mineralized tissues and is subjected to variety of posttranslational modifications including phosphorylation and glycosylation. We have expressed the full-length and half domains of bovine bone sialoprotein in a prokaryotic system and identified the phosphorylation sites of casein kinase II. The N-terminal automated solid-phase sequencing defined four phosphorylated peptides: residues 28-38 (LEDSPEENGVFK), 51-86 (FYPELKRFAVQSSSPDSPSPEENGNGDSPSPEEEEEEEETSP), 151-165 (EDESPDEEEEEEEEEE), and 295-305 (GRGYDSPYDGQD). Nine phosphoserines were identified within the four peptides. Seven of them were in the N-terminus (S31, S64, S66, S67, S75, S76, and S86) and two were in the C-terminus (S154 and S300) of the protein.