Lorentsen Rikke H, Møller Charlotte H, Etzerodt Michael, Thøgersen Hans C, Holtet Thor L
Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark.
Org Biomol Chem. 2003 May 21;1(10):1657-63. doi: 10.1039/b210149a.
A library of blood coagulation factor Xa (FXa)-trypsin hybrid proteases was generated and displayed on phage for selection of derivatives with the domain "architecture" of trypsin and the specificity of FXa. Selection based on binding to soybean trypsin inhibitor only provided enzymatically inactive derivatives, due to a specific mutation of serine 195 of the catalytic triad to a glycine, revealing a significant selection pressure for proteolytic inactive derivatives. By including a FXa peptide substrate in the selection mixture, the majority of the clones had retained serine at position 195 and were enzymatically active after selection. Further, with the inclusion of bovine pancreatic trypsin inhibitor, in addition to the peptide substrate, the selected clones also retained FXa specificity after selection. This demonstrates that affinity selection combined with appropriate deselection provides a simple strategy for selection of enzyme derivatives that catalyse a specific reaction.
构建了一个凝血因子Xa（FXa）-胰蛋白酶杂合蛋白酶文库，并展示在噬菌体上，以筛选具有胰蛋白酶“结构域”结构和FXa特异性的衍生物。基于与大豆胰蛋白酶抑制剂的结合进行筛选，仅得到了酶活性缺失的衍生物，这是由于催化三联体中丝氨酸195特异性突变为甘氨酸所致，这揭示了对蛋白水解无活性衍生物的显著选择压力。通过在筛选混合物中加入FXa肽底物，大多数克隆在195位保留了丝氨酸，筛选后具有酶活性。此外，除了肽底物外，加入牛胰蛋白酶抑制剂后，所选克隆在筛选后也保留了FXa特异性。这表明亲和选择与适当的反选择相结合，为筛选催化特定反应的酶衍生物提供了一种简单的策略。
