AEGiS-11IAC: Separation of gp120 and gp160 by Cibacron Blue 3GA.

11th International AIDS Conference


Vancouver, British Columbia — July 7-12, 1996

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Separation of gp120 and gp160 by Cibacron Blue 3GA.

Int Conf AIDS 1996 Jul 7-12; 11:56 (abstract no. Mo.A.1006)
Hattori T, Sato Y, Kubo T, Zhan X, Sakaida H, Nishikawa S, Uchiyama T; Institute for Virus Research, Kyoto Univer., Kyoto, Japan. Tel/Fax: 81-75-751-3986. E-mail: thattori@virusl.virus.kyoto-u.ac.jp.

OBJECTIVES: We have attempted to separate gp120 from gp160 from the culture supernatants of gp120/gp160 secreting CHO cells to characterize the biological and biochemical features of both proteins.

METHODS: CHO-SEC cells were used for secreting cells (kindly provided from Dr. Weiss at FDA). The cells are made by transfecting HIV-1 (HXB-2) envelope gene (5999-8270). The cells secrete transmembrane-portion-truncated gp160 and gp120 in culture medium. A large scale culture of CHO-SEC cells using GCM#001 medium (RPMI based synthetic medium containing 1 mg/ml human serum albumin) was performed using collagen micro sphere (VERAX System One) for 4 weeks. gp120/gp160 was detected by Western blotting using anti-V3 loop monoclonal antibody 902(NIH AIDS Research and Reference Reagent Program). After concentration of the culture supernatants by centriplus 100(M.W. Cut-off 105), the samples were applied to Cibacron-Blue-F-3GA coupled column (Hi-Trap) and analyzed by HPLC.

RESULTS: The approximate amounts of gp120/160 in the obtained culture supernatants were 1 microgram/ml before concentration. Fifty times concentrated culture supernatants was applied onto 1ml Hi-Trap column in 50 mM potassium phosphate buffer containing 0.1M KCl. The proteins were eluted by a step-wise increase of KCl concentration (0.1M-2M). The band corresponding to gp120 was detected in a lower KCl concentration (0.5-1.0 M), on the other hand gp160 was detected in higher concentration of KCl(0.9-1.5 M). Albumin was detected in lower KCl concentration fractions.

CONCLUSION: Cibacron blue 3GA is a useful ligand to separate gp120 and gp160. Further analysis of the binding of the dye to gp120/gp160 is in progress.

Keywords: AEGIS, Triazines, HIV-1, Acquired Immunodeficiency Syndrome, Anti-HIV Agents, Dyes, Antibodies, Monoclonal, Cibacron Blue F 3GA, Human, ethnology, ICA11KWDaegis,triazines,hiv-1,acquiredimmunodeficiencysyndrome,anti-hivagents,dyes,antibodies,monoclonal,cibacronbluef3ga,human,ethnology,ica11

960707
MoA1006

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