Purification and characterization of simian immunodeficiency virus (SIVmac) envelope glycoprotein gp130 from virus-infected cells.
A non-denaturing method has been developed for the purification of the envelope glycoprotein gp130 of the simian immunodeficiency virus (SIV) using infected cells as starting material. The procedure involves solubilization of cells infected with SIV (SIVmac251), enrichment of glycoproteins by lectin affinity chromatography, fractionation by reverse phase chromatography and purification by immunoaffinity chromatography. This procedure results in a greater than 95% purification of gp130 as assessed by polyacrylamide gel electrophoresis. There is no evidence for the presence of other virus-derived proteins after Western blot analysis using antibodies specific for virus proteins. Lectin-binding studies suggest that carbohydrate groups on the infected-cell-derived gp130 may differ from those on recombinant counterparts expressed in Chinese hamster ovary cells and Baculovirus-infected insect cells. The purified gp130 is highly immunogenic in rabbits and maintains the capacity to bind the CD4 receptor. A sufficient quantity of the infected-cell-derived gp130 has been prepared for immunization studies and subsequent live virus challenge studies in macaques.
Jones
D H
DH
Division of Pathology, Centre for Applied Microbiology & Research, Salisbury, Wiltshire, UK.
McBride
B W
BW
Roff
M A
MA
Maloney
V
V
Farrar
G H
GH
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D015704:CD4 Antigens / Q000378:metabolism; D016466:CHO Cells; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D015686:Gene Products, env / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D037102:Lectins / Q000378:metabolism; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D009036:Moths; D011817:Rabbits; D015302:Simian Immunodeficiency Virus
D015704:CD4 Antigens; D015686:Gene Products, env; D037102:Lectins; C078684:simian immunodeficiency virus gp130