Document Detail


Inhibition of V3-specific cleavage of recombinant HIV-1 gp120 produced in Chinese hamster ovary cells.
MedLine Citation:
PMID:  18406166     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Sean X Du; Li Xu; Sridhar Viswanathan; Robert G Whalen
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-02-21
Journal Detail:
Title:  Protein expression and purification     Volume:  59     ISSN:  1096-0279     ISO Abbreviation:  Protein Expr. Purif.     Publication Date:  2008 Jun 
Date Detail:
Created Date:  2008-05-06     Completed Date:  2008-06-06     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9101496     Medline TA:  Protein Expr Purif     Country:  United States    
Other Details:
Languages:  eng     Pagination:  223-31     Citation Subset:  IM    
Affiliation:
Maxygen, Inc., Infectious Diseases, 515 Galveston Drive, Redwood City, CA 94063, USA.
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MeSH Terms
Descriptor/Qualifier:
AIDS Vaccines
Amino Acid Sequence
Animals
CHO Cells
Chromatography, Affinity
Cricetinae
Cricetulus
Culture Media
HIV Envelope Protein gp120 / chemistry,  isolation & purification*,  metabolism*
HIV-1*
Humans
Molecular Sequence Data
Peptide Fragments / chemistry,  isolation & purification,  metabolism
Protease Inhibitors / pharmacology
Protein Structure, Tertiary
Recombinant Proteins / chemistry,  isolation & purification*,  metabolism*
Trypsin / drug effects,  metabolism
Chemical
Reg. No./Substance:
0/AIDS Vaccines; 0/Culture Media; 0/HIV Envelope Protein gp120; 0/Peptide Fragments; 0/Protease Inhibitors; 0/Recombinant Proteins; EC 3.4.21.4/Trypsin

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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