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

被引:68
作者
Zhang, CWH
Chishti, Y
Hussey, RE
Reinherz, EL
机构
[1] Dana Farber Canc Inst, Immunobiol Lab, Boston, MA 02115 USA
[2] Harvard Univ, Sch Med, Dept Med, Boston, MA 02115 USA
关键词
D O I
10.1074/jbc.M107147200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Efforts to understand the molecular basis of human immunodeficiency virus (HM 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) (K-D = 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-SrV mac32H implies quaternary structural differences in ground states of their respective spike proteins.
引用
收藏
页码:39577 / 39585
页数:9
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