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Originally published In Press as doi:10.1074/jbc.M107147200 on August 20, 2001

J. Biol. Chem., Vol. 276, Issue 43, 39577-39585, October 26, 2001
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Expression, Purification, and Characterization of Recombinant HIV gp140
THE gp41 ECTODOMAIN OF HIV OR SIMIAN IMMUNODEFICIENCY VIRUS IS SUFFICIENT TO MAINTAIN THE RETROVIRAL ENVELOPE GLYCOPROTEIN AS A TRIMER*

Catherine W-H. Zhang, Yasmin Chishti, Rebecca E. Hussey, and Ellis L. ReinherzDagger

From the Laboratory of Immunobiology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachussetts 02115

Efforts to understand the molecular basis of human immunodeficiency virus (HIV) envelope glycoprotein function have been hampered by the inability to generate sufficient quantities of homogeneous material. We now report on the high level expression, purification, and characterization of soluble HIV gp140 ectodomain proteins in Chinese hamster ovary-Lec3.2.8.1 cells. Gel filtration and analytical ultracentrifugation show that the uncleaved ADA strain-derived gp140 proteins are trimeric without further modification required to maintain oligomers. These spike proteins are native as judged by soluble CD4 (sCD4) (KD = 1-2 nM) and monoclonal antibody binding studies using surface plasmon resonance. CD4 ligation induces conformational change in the trimer, exposing the chemokine receptor binding site as assessed by 17b monoclonal antibody reactivity. Lack of anti-cooperativity in sCD4-ADA trimer interaction distinct from that observed with sCD4-SIV mac32H implies quaternary structural differences in ground states of their respective spike proteins.

* This work was supported by National Institutes of Health Grant AI43649.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Laboratory of Immunobiology, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3412; Fax: 617-632-3351; E-mail: ellis_reinherz@dfci.harvard.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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