Hansen J, Billich S, Schulze T, Sukrow S, Moelling K
Max-Planck-Institut für Molekulare Genetik, Abt. Schuster, Berlin, FRG.
EMBO J. 1988 Jun;7(6):1785-91. doi: 10.1002/j.1460-2075.1988.tb03009.x.
Retroviruses code for a specific protease which is essential for polyprotein precursor processing and viral infectivity. The HIV-specific protease has been predicted to be an aspartic protease which is located at the amino terminus of the pol gene. We have prepared several constructs for bacterial expression of the protease. Two of them span the whole protease region and result in its autocatalytic activation. Analysis of the dynamics of this activation indicates a two-step process which starts at the carboxy terminus and ends at the amino terminus of the protease. The activated protease is a molecule of 9 kd as evidenced by monoclonal antibody in immunoblot analysis. A construct in which the carboxy terminus of the protease is deleted results in a stable, enzymatically inactive 27-kd protein which proved useful as substrate since it contains one of the predicted cleavage sites. The stability of this protein indicates that the carboxy-terminal sequences of the protease are essential for its activity and its autocatalytic activation. The protease which is very hydrophobic was solubilized by acetone treatment and passaged over ultrogel and propylagarose columns for partial purification. It elutes as a dimer and tends to aggregate. It is inhibited by pepstatin A in agreement with its expected active site and its theoretical classification as aspartic protease. Cleavage of the gag precursor results in the mature capsid protein, p17. The protease does not, however, cleave the denatured 27-kd substrate or the denatured gag precursor. Therefore its specificity appears to be not solely sequence- but also conformation-dependent. This property needs to be taken into account for the development of protease inhibitors for therapy of AIDS.
逆转录病毒编码一种特定的蛋白酶，该蛋白酶对于多蛋白前体的加工和病毒感染性至关重要。据预测，HIV特异性蛋白酶是一种天冬氨酸蛋白酶，位于pol基因的氨基末端。我们制备了几种用于该蛋白酶细菌表达的构建体。其中两种跨越整个蛋白酶区域，并导致其自催化激活。对这种激活动力学的分析表明这是一个两步过程，从蛋白酶的羧基末端开始，到氨基末端结束。免疫印迹分析中，单克隆抗体证明激活后的蛋白酶是一个9kd的分子。一种缺失蛋白酶羧基末端的构建体产生了一种稳定的、无酶活性的27kd蛋白质，由于它包含一个预测的切割位点，因此被证明可作为底物。这种蛋白质的稳定性表明蛋白酶的羧基末端序列对其活性和自催化激活至关重要。这种非常疏水的蛋白酶通过丙酮处理溶解，并通过超凝胶和丙基琼脂糖柱进行部分纯化。它以二聚体形式洗脱并倾向于聚集。它被胃蛋白酶抑制剂A抑制，这与其预期的活性位点以及作为天冬氨酸蛋白酶的理论分类一致。gag前体的切割产生成熟的衣壳蛋白p17。然而，该蛋白酶不会切割变性的27kd底物或变性的gag前体。因此，其特异性似乎不仅取决于序列，还取决于构象。在开发用于治疗艾滋病的蛋白酶抑制剂时需要考虑这一特性。
