Expression and purification of recombinant human SPARC produced by baculovirus.
SPARC (secreted protein acidic and rich in cysteine/osteonectin/BM-40), a matrix-associated protein, disrupts cell adhesion and inhibits the proliferation of many cultured cells. We report the expression of recombinant human protein (rhSPARC) in a baculovirus expression system. This procedure routinely yields approximately 1 mg of purified protein per 500 ml of culture supernate. rhSPARC produced by insect cells migrates at the appropriate molecular weight under reducing and nonreducing conditions. The rhSPARC purified from insect cell media appeared structurally similar to SPARC purified from mammalian tissue culture by the criterion of circular dichroism. In addition, a series of anti-SPARC and anti-SPARC peptide antibodies recognized insect cell rhSPARC. We also show that rhSPARC produced in this system is glycosylated and is biologically active, as assessed by inhibition of endothelial cell proliferation and induction of collagen I mRNA in mesangial cells. Significant amounts of rhSPARC can now be generated in the absence of contaminating mammalian proteins for structure/function assays of SPARC activities.
Bradshaw
A D
AD
Department of Vascular Biology, Hope Heart Institute, Seattle, Washington, 98104, USA.
Bassuk
J A
JA
Francki
A
A
Sage
E H
EH
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002118:Calcium / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002942:Circular Dichroism; D003094:Collagen / Q000235:genetics; D016895:Culture Media, Serum-Free; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects; D006031:Glycosylation; D006801:Humans; D008970:Molecular Weight; D015676:Osteonectin / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D017433:Protein Structure, Secondary / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D018411:Spodoptera; D015533:Transcriptional Activation / Q000187:drug effects
D016895:Culture Media, Serum-Free; D015676:Osteonectin; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003094:Collagen; D002118:Calcium