Nonaffinity purification of recombinant gp120 for use in AIDS vaccine development.
The gene encoding the major envelope glycoprotein of the HIV-SF2 isolate was engineered for the secretion of recombinant gp120 (rgp120SF2) from permanent Chinese hamster ovary (CHO) cell lines. Cellular production methods were scaled up and a method for purification of the secreted glycoprotein was devised. Mild purification conditions were selected in order to preserve the native structure of the protein. rgp120SF2 exhibits a molecular weight of 120 kDa in reduced or nonreduced SDS gels; thus the polypeptide chain is intact. Deglycosylated rgp120SF2 has the predicted molecular weight of the polypeptide backbone, 54 kDa. Gel-filtration HPLC in a nondenaturing buffer at neutral pH yields a molecular weight estimate of approximately 120 kDa. Purified rgp120 closely resembles authentic viral gp120 by several physical, chemical, and immunochemical tests. rgp120SF2 reacts strongly with human HIV-positive sera, monoclonal antibodies reactive with HIV-SF2 and HIV-MN viral envelope, and a human virus-neutralizing monoclonal antibody that maps to a conserved discontinuous epitope on HIV-1 gp120. Purified rgp120SF2 forms a 1:1 molecular complex with soluble recombinant human CD4 (rCD4) receptor, as demonstrated by gel-filtration HPLC; binding is high affinity (Kd approximately 2 x 10(-9) M).
Scandella
C J
CJ
Department of Process Development, Chiron Corporation, Emeryville, California 94608.
Kilpatrick
J
J
Lidster
W
W
Parker
C
C
Moore
J P
JP
Moore
G K
GK
Mann
K A
KA
Brown
P
P
Coates
S
S
Chapman
B
B
D016915:AIDS Vaccines / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D006224:Cricetinae; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D015497:HIV-1 / Q000235:genetics / Q000276:immunology*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D016915:AIDS Vaccines; D000596:Amino Acids; D015704:CD4 Antigens; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D011994:Recombinant Proteins