Directed evolution of three-finger toxin to produce serine protease inhibitors
Directed evolution of three-finger toxin to produce serine protease inhibitors
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DOI：
10.3109/10799893.2013.865747
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发表时间：
2014-06-01
影响因子：
2.8
通讯作者：
Kubo, Tai
中科院分区：
文献类型：
--
作者：
Cai, Weiyan;Naimuddin, Mohammed;Kubo, Tai
定向进化是一种非常流行的策略，用于改善生物物理性质，甚至用于产生具有新功能的蛋白质。组合蛋白质工程的最新进展意味着，现在有可能开发出可以取代整个抗体相关特性的蛋白质支架，成为新兴的治疗性蛋白质。特别是，富含二硫键的蛋白质是寻找新分子的定向进化的有吸引力的模板，因为它们可以调节受体、酶和其他分子的活动。在此之前，我们已经证明了针对白介素6受体(IL-6R)的功能调节分子可以通过三指毒素(3F)支架的定向进化而获得。本研究选择胰酶作为定向进化的靶标，以进一步探索3F文库的潜在用途。经过七轮选择，DNA序列收敛。筛选出的重组蛋白对胰酶具有抑制活性(K-I为33-450 nM)。6组中有3组的K-I值与牛胰酶抑制剂和大豆胰蛋白酶抑制剂相当。其中两个候选药物对胰凝乳酶和激肽释放酶也有抑制作用。本研究表明，3F蛋白适合于制备高多样性文库，可用于获得蛋白水解酶抑制剂。除了我们之前成功的靶向IL-6R之外，我们研究中开发的技术可能在生成感兴趣的靶点的调节分子方面有广泛的应用，如受体、用于研究的酶、诊断应用和治疗用途。
Directed evolution is a very popular strategy for improving biophysical properties and even for generating proteins with novel functions. Recent advances in combinatorial protein engineering mean it is now possible to develop protein scaffolds that could substitute for whole antibody-associated properties as emerging therapeutic proteins. In particular, disulfide-rich proteins are attractive templates for directed evolution in the search for novel molecules because they can regulate the activities of receptors, enzymes, and other molecules. Previously, we demonstrated that functional regulatory molecules against interleukin-6 receptor (IL-6R) could be obtained by directed evolution of the three-finger toxin (3F) scaffold. In the present study, trypsin was selected as a target for directed evolution to further explore the potential use of the 3F cDNA display library. After seven rounds of selection, the DNA sequences converged. The recombinant proteins produced by the selected candidates had inhibitory activity against trypsin (K-i of 33-450 nM). Three of the six groups had K-i values that were comparable to bovine pancreatic trypsin inhibitor and soybean trypsin inhibitor. Two of the candidates also had inhibitory effects against chymotrypsin and kallikrein. This study suggests that 3F protein is suitable for the preparation of high-diversity libraries that can be utilized to obtain protease inhibitors. In addition to our previous successful targeting of IL-6R, the technique developed in our studies may have wide applications in the generation of regulatory molecules for targets of interest, such as receptors, enzymes for research, diagnostic applications, and therapeutic uses.