Expression, purification, and functional testing of recombinant CYR61/CCN1

The human cysteine-rich protein 61 (CYR61/CCN1) belongs to the CCN family of genes which plays an important role in cellular processes such as proliferation, migration, adhesion, and differentiation. These extracellular matrix signaling molecules consist of a modular structure and contain 38 conserved cysteine residues. Previously, we have shown that CYR61 is expressed in human osteoblasts and is regulated by bone-relevant growth factors. The protein also plays a role in angiogenesis. The open reading frame was cloned into a baculovirus expression vector and transfected into SF-21 insect cells. Recombinant protein was expressed as a fusion protein with the Fc-domain of human IgG and purified using affinity chromatography on protein G-Sepharose columns. The chorioallantoic membrane assay verified that blood vessel formation was stimulated by rCYR61. Additionally, human primary mesenchymal stem cells, osteoblasts, and endothelial cells responded to CYR61 treatment by a markedly stimulated proliferation. rCYR61-Fc represents a tool to elucidate its role in cells of the bone microenvironment.