title: Expression of the glycosylphosphatidylinositol-linked complement-inhibiting protein CD59 antigen in insect cells using a baculovirus vector.
abstract: CD59 antigen (CD59) is a glycosylphosphatidylinositol (GPI)-linked membrane glycoprotein which protects human cells from complement-mediated lysis. Here we report the expression of functionally active CD59 in Spodoptera frugiperda insect cells using a baculovirus vector. Recombinant CD59 was expressed abundantly on the surface of the insect cells and protected the cells from lysis by human complement. The protein was released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C, indicating that it was attached to the insect cell membrane via a GPI anchor. The cells also secreted CD59 into the culture medium. Recombinant CD59 was affinity-purified from spent culture medium and from detergent extract of transfected cells. Protein purified from both sources produced multiple bands on SDS/PAGE, all of a lower apparent molecular mass than the human erythrocyte protein. However, N-terminal protein sequencing and deglycosylation studies confirmed that signals for leader peptide cleavage and N-linked glycosylation had been recognized in the insect cells, suggesting that the differences in apparent molecular mass between the native and recombinant proteins were attributable to the extent of glycosylation. Protein derived from both sources was, in part, GPI-anchored as demonstrated by phase-partition studies and incorporation into cells membranes. Incorporated recombinant protein rendered erythrocytes resistant to complement lysis.
authors.affiliation: Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, U.K.
authors.forename: A
authors.initials: A
authors.lastname: Davies
authors.forename: B P
authors.initials: BP
authors.lastname: Morgan
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics*; D018957:CD59 Antigens; D002460:Cell Line; D003165:Complement System Proteins / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000032:analysis / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D008562:Membrane Glycoproteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D009036:Moths / Q000378:metabolism*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism
chemical_list: D015703:Antigens, CD; D018957:CD59 Antigens; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D003165:Complement System Proteins; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase