Expression of a recombinant baculovirus for vitronectin in insect cells: purification, characterization of post-translational modifications and functional studies of the recombinant protein.
Vitronectin (VN) is a cell attachment glycoprotein present in plasma and the extracellular matrix that also has multiple regulatory roles in the complement, coagulation, and fibrinolytic systems. VN stabilizes plasminogen activator inhibitor-1 (PAI-1) with potential binding sites reported at both the somatomedin B and heparin binding domains. In order to perform detailed structure-function analyses of VN, we have produced and expressed a recombinant baculovirus containing the cDNA of human VN. Recombinant VN (rVN) was secreted as a single chain 70-kDa protein into the serum-free culture medium of infected Spodoptera frugiperda cells. Maximum rVN levels of 33.9 mg/liter were obtained 48 hours post-infection. Purification of rVN from serum-free medium was achieved with single-step anion-exchange chromatography. Several native post-translational modifications were demonstrated in rVN including the presence of N-linked oligosaccharides, tyrosine sulfation, and serine phosphorylation. Recombinant VN retained cell and PAI-1 binding capacity. Therefore, rVN expressed for the first time in insect cells retains many post-translational modifications and functional activities and will be useful for additional structure-function analyses.
Zhao
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Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Sane
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D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002448:Cell Adhesion; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology; D010766:Phosphorylation; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011994:Recombinant Proteins / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology; D019096:Vitronectin
D006023:Glycoproteins; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D019096:Vitronectin; D014415:Tunicamycin