Purpose of Work We have developed a strategy of designing multi-functional extracellular matrix proteins for functionalizing bone tissue engineering scaffolds and other biomedical surfaces to achieve improvements in bone grafting, bone repair and bone regeneration. We developed a novel extracellular matrix protein designed to have a cell adhesive RGD sequence derived from fibronectin and active functional unit of osteocalcin (OC) containing Ca2(+)-binding sites for immobilization to mineral component of bone, hydroxyapatite (HA). The fusion protein, designated FNRGD/OC, was expressed in Escherichia coli and purified with affinity chromatography using a His-tag. The resultant FNRGD/OC fusion protein preferentially bound to HA, promoted cell adhesive activity, and stimulated differentiation of MC3T3-E1 cell.