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117 A Chimeric Protein of SIV Envelope Glycoprotein Gp-160e and E. coli Aspartate Transcarbamoylase
B. Chen*1, J.J. Skehel3, E.L. Reinherz4, S.C. Harrison1,2, and D.C. Wiley1,2
1The Children's Hosp./Harvard Univ., Boston, MA, USA; 2 Howard Hughes Med. Inst., Boston, MA, USA; 3Natl. Inst. for Med. Res., London, UK; and 4Dana Farber Cancer Inst., Boston, MA, USA
Background: The envelope glycoproteins of the human immunodeficiency virus (HIV) and the related simian immunodeficiency virus (SIV) mediate viral entry into host cells by fusing the viral membrane with the target cell membrane.
Methods:Recently, we have reported expression, purification, and characterization of gp160e, the soluble, trimeric ectodomain of the SIV envelope glycoprotein, gp-160. Here, we have expressed and purified chimeric proteins of SIV gp160e and its variants with the catalytic subunit (C) of E. coli ATCase (aspartate transcarbamoylase) to facilitate structural studies of the envelope glycoprotein. Results:The fusion proteins bind viral receptor CD4 and a number of monoclonal antibodies specific for SIV gp160e. The chimeric molecule also has ATCase activity, which requires trimerization of the ATCase C chains, suggesting they are properly folded and that the whole fusion protein is trimeric. When ATCase regulatory subunit dimers (R2) are added, the fusion protein assembles into dimers of trimers as expected from the structure of the C6R6 ATCase.
Conclusions:The production of the fusion proteins may enhance the possibilities for structure determination of the envelope glycoprotein by either cryo-electron microscopy or x-ray crystallography.
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