Abstract
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) (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-SIV mac32H implies quaternary structural differences in ground states of their respective spike proteins.
Highlights
human immunodeficiency virus (HIV) infection requires the serial interaction of its envelope proteins with both a cellular receptor and co-receptor (9 –13)
We report here the expression and purification of stable trimeric gp140 of the macrophage-tropic, CCR5-dependent ADA strain of HIV [35, 36] and an ADA/Simian immunodeficiency virus (SIV) chimera produced in a Lec3.2.8.1 derivative of Chinese hamster ovary (CHO) cells
Stable Expression of Soluble gp140 Protein in CHOLec3.2.8.1 Cell Lines—In order to produce soluble trimeric HIV or HIV/SIV chimeric envelope proteins by recombinant eukaryotic expression, four constructs were made in pEE-14 (Fig. 1)
Summary
Gp140 Expression Constructs—The pEE14-GS vector [37] containing a Kozak sequence and encoding the tissue plasminogen activator (tPA) leader peptide in lieu of the endogenous viral sequence upstream of the mature gp140 coding sequence was used for all expression constructs. Analytical Ultracentrifugation—The affinity-purified and sized gp140ADAC1 and gp140ADA/SIV proteins in PBS were analyzed by centrifugation at speeds of 6000 and 8000 rpm on a Beckman XL-A analytical ultracentrifugation at 4 °C The concentrations of both proteins were 0.19 and 0.35 mg/ml, respectively, with the data fitted to a singlespecies model. Deglycosylation of the Trimeric gp140 Proteins—Trimeric gp140 proteins were deglycosylated by endoglycosidase H (Roche Molecular Biochemicals) (1 mg of protein digested by 250 milliunits of endoglycosidase H) at 30 °C for 6 h in 50 mM NaAc, pH 6.0, in the presence or absence of Fab fragments of the indicated mAbs. The deglycosylated protein was analyzed by SDS-PAGE and gel filtration chromatography using an analytic Superdex 200 column on an AKTA FPLC (Amersham Pharmacia Biotech)
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