Purification of biologically active SPARC expressed in Saccharomyces cerevisiae.
SPARC (secreted protein, acidic and rich in cysteine) is a secreted, Ca+2-binding glycoprotein that modulates interactions between cells and their immediate extracellular matrix. Traditional sources of SPARC have been mammalian bone, platelets, a basement membrane tumor, and cultured cells; most if not all preparations, however, contain platelet-derived growth factor and one or more serum proteins that bind specifically to purified SPARC. To avoid these contaminants, as well as the toxic lipid moiety associated with endotoxin, we expressed recombinant wild-type and a mutated murine SPARC in two strains of Saccharomyces cerevisiae: one strain was transfected with an expression vector encoding a proprietory signal peptide that directed the secretion of the recombinant protein. Recombinant SPARC was also purified from cell lysates of a different, nonreverting strain of S. cerevisiae that was optimized for large-scale fermentation runs. A mutant murine SPARC lacking the single glycosylation site was also expressed following substitution of Asn98 with Asp98 in the wild-type sequence. Purification of SPARC was achieved by copper-affinity and hydrophobic-interaction chromatography. Both the wild-type and the glycosylation-defective recombinant proteins exhibited high levels of activity in two bioassays with endothelial cells: inhibition of cell spreading/disruption of actin microfilaments and competition for the binding of nonrecombinant 125I-labeled SPARC to the cell surface. The availability of biologically active, recombinant SPARC will facilitate investigation of the structural and functional properties of this protein, which is expressed at high levels in healing wounds, atherosclerotic plaque, and several cancers and diseases of connective tissue.
Yost
J C
JC
Department of Biological Structure, University of Washington, Seattle 98195.
Bell
A
A
Seale
R
R
Sage
E H
EH
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D002845:Chromatography; D004730:Endothelium, Vascular / Q000187:drug effects / Q000648:ultrastructure; D015870:Gene Expression; D006031:Glycosylation; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015676:Osteonectin / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014162:Transfection
D000199:Actins; D015676:Osteonectin; D011994:Recombinant Proteins