Scandella C J, Kilpatrick J, Lidster W, Parker C, Moore J P, Moore G K, Mann K A, Brown P, Coates S, Chapman B
Department of Process Development, Chiron Corporation, Emeryville, California 94608.
AIDS Res Hum Retroviruses. 1993 Dec;9(12):1233-44. doi: 10.1089/aid.1993.9.1233.
The gene encoding the major envelope glycoprotein of the HIV-SF2 isolate was engineered for the secretion of recombinant gp120 (rgp120SF2) from permanent Chinese hamster ovary (CHO) cell lines. Cellular production methods were scaled up and a method for purification of the secreted glycoprotein was devised. Mild purification conditions were selected in order to preserve the native structure of the protein. rgp120SF2 exhibits a molecular weight of 120 kDa in reduced or nonreduced SDS gels; thus the polypeptide chain is intact. Deglycosylated rgp120SF2 has the predicted molecular weight of the polypeptide backbone, 54 kDa. Gel-filtration HPLC in a nondenaturing buffer at neutral pH yields a molecular weight estimate of approximately 120 kDa. Purified rgp120 closely resembles authentic viral gp120 by several physical, chemical, and immunochemical tests. rgp120SF2 reacts strongly with human HIV-positive sera, monoclonal antibodies reactive with HIV-SF2 and HIV-MN viral envelope, and a human virus-neutralizing monoclonal antibody that maps to a conserved discontinuous epitope on HIV-1 gp120. Purified rgp120SF2 forms a 1:1 molecular complex with soluble recombinant human CD4 (rCD4) receptor, as demonstrated by gel-filtration HPLC; binding is high affinity (Kd approximately 2 x 10(-9) M).
对编码HIV - SF2分离株主要包膜糖蛋白的基因进行了改造，以便从中国仓鼠卵巢（CHO）永久细胞系中分泌重组gp120（rgp120SF2）。扩大了细胞生产方法的规模，并设计了一种分泌糖蛋白的纯化方法。选择温和的纯化条件以保留蛋白质的天然结构。在还原或非还原SDS凝胶中，rgp120SF2的分子量为120 kDa；因此多肽链是完整的。去糖基化的rgp120SF2具有多肽主链的预测分子量，即54 kDa。在中性pH的非变性缓冲液中进行凝胶过滤HPLC，得出的分子量估计约为120 kDa。通过多项物理、化学和免疫化学测试，纯化的rgp120与天然病毒gp120非常相似。rgp120SF2与人类HIV阳性血清、与HIV - SF2和HIV - MN病毒包膜反应的单克隆抗体以及一种针对HIV - 1 gp120上保守的不连续表位的人类病毒中和单克隆抗体强烈反应。如凝胶过滤HPLC所示，纯化的rgp120SF2与可溶性重组人CD4（rCD4）受体形成1:1分子复合物；结合具有高亲和力（解离常数约为2×10⁻⁹ M）。
