title: Inhibition of V3-specific cleavage of recombinant HIV-1 gp120 produced in Chinese hamster ovary cells.
abstract: Specific proteolytic cleavage of the gp120 subunit of the HIV-1 envelope (Env) glycoprotein in the third variable domain (V3) has previously been reported to occur in several cell lines, including Chinese hamster ovary cells that have been used for production of Env-based HIV vaccine candidates. Here we report that this proteolytic activity on JRCSF gp120 is dependent on cell density, medium conditions, and supernatant concentration. The resulting cleaved polypeptides cannot be separated from intact gp120 by conventional or affinity chromatography under non-reducing conditions. Inhibitor studies reveal that Pefabloc and benzamidine, but not chymostatin, block gp120 cleavage in a dose-dependent fashion, suggesting the presence of a trypsin-like serine protease in CHO-K1 cells. The proteolytic activity is increased with certain types of cell culture growth media. A combination of serum-free OptiMEM media during expression and potent protease inhibitors post-expression can effectively prevent HIV gp120 degradation. The same strategy can be applied to the expression and purification of gp120 of other strains or other forms of envelope-based vaccine candidates containing V3 sequences.
authors.affiliation: Maxygen, Inc., Infectious Diseases, 515 Galveston Drive, Redwood City, CA 94063, USA.
authors.forename: Sean X
authors.initials: SX
authors.lastname: Du
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.forename: Sridhar
authors.initials: S
authors.lastname: Viswanathan
authors.forename: Robert G
authors.initials: RG
authors.lastname: Whalen
mesh_terms: D016915:AIDS Vaccines; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D015497:HIV-1; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D014357:Trypsin / Q000187:drug effects / Q000378:metabolism
chemical_list: D016915:AIDS Vaccines; D003470:Culture Media; D015699:HIV Envelope Protein gp120; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D014357:Trypsin