PURIFICATION OF BIOLOGICALLY-ACTIVE SPARC EXPRESSED IN SACCHAROMYCES-CEREVISIAE

SPARC (secreted protein, acidic and rich in cysteine) is a secreted, C a+2-binding glycoprotein that modulates interactions between cells and their immediate extracellular matrix. Traditional sources of SPARC ha ve been mammalian bone, platelets, a basement membrane tumor, and cult ured cells; most if not all preparations, however, contain platelet-de rived growth factor and one or more serum proteins that bind specifica lly to purified SPARC. To avoid these contaminants, as well as the tox ic lipid moiety associated with endotoxin, we expressed recombinant wi ld-type and a mutated murine SPARC in two strains of Saccharomyces cer evisiae: one strain was transfected with an expression vector encoding a proprietory signal peptide that directed the secretion of the recom binant protein. Recombinant SPARC was also purified from cell lysates of a different, nonreverting strain of S. cerevisiae that was optimize d for large-scale fermentation runs. A mutant murine SPARC lacking the single glycosylation site was also expressed following substitution o f Asn(98) with Asp(98) in the wild-type sequence. Purification of SPAR C was achieved by copper-affinity and hydrophobic-interaction chromato graphy. Both the wild-type and the glycosylation-defective recombinant proteins exhibited high levels of activity in two bioassays with endo thelial cells: inhibition of cell spreading/disruption of actin microf ilaments and competition for the binding of nonrecombinant I-125-label ed 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 lev els in healing wounds, atherosclerotic plaque, and several cancers and diseases of connective tissue. (C) 1994 Academic Press, Inc.