CD4-affinity purification of recombinant and native HIV gp120 and comparison of the affinity constants for the receptor.
Soluble CD4-immunoglobulin chimeric proteins were covalently attached to CNBr-activated Sepharose. This affinity matrix was used to establish a powerful new method to isolate different species of the HIV external glycoprotein gp120 from cell-free culture supernatants. Recombinant gp120 was expressed in Baculovirus-infected insect cells and isolated from cell-free culture supernatants. The recombinant protein has an apparent molecular mass of 130 kDa. These two gp120 species were shown to be of identical molecular size after complete deglycosylation achieved by endoglycosidase treatment, and they bound to CD4-H gamma l with the same binding constant, that was reported for native forms of gp120 and CD4. Thus the different glycosylation of gp120 does not influence its affinity to CD4 and the gp120-CD4 complex can be reversibly dissociated.
Moritz
D
D
Central Research Units, F. Hoffman-La Roche Ltd., Basel, Switzerland.
Dirckx
L
L
Mous
J
J
Schneider
J
J
D015153:Blotting, Western; D015704:CD4 Antigens / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D015699:HIV Envelope Protein gp120 / Q000302:isolation & purification* / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D011994:Recombinant Proteins