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.
Schütze
Norbert
N
Orthopaedic Department, Molecular Orthopaedics, University of Würzburg, Germany. n-schuetze.klh@mail.uni-wuerzburg.de
Kunzi-Rapp
Karin
K
Wagemanns
Rita
R
Nöth
Ulrich
U
Jatzke
Susanne
S
Jakob
Franz
F
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D049033:Chorioallantoic Membrane / Q000098:blood supply / Q000187:drug effects; D055515:Cysteine-Rich Protein 61; D004591:Electrophoresis, Polyacrylamide Gel; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D015870:Gene Expression / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000235:genetics; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects; D008969:Molecular Sequence Data; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D018411:Spodoptera; D014162:Transfection
C501848:CCN1 protein, human; D055515:Cysteine-Rich Protein 61; D017874:Immediate-Early Proteins; D007141:Immunoglobulin Fc Fragments; D036341:Intercellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins