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首页> 外文期刊>Journal of Molecular Biology >Discovery of improved EGF agonists using a novel in vitro screening platform.
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Discovery of improved EGF agonists using a novel in vitro screening platform.

机译：使用新型体外筛选平台发现改良的EGF激动剂。

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摘要

Directed evolution is a powerful strategy for protein engineering; however, evolution of pharmaceutical proteins has been limited by the reliance of current screens on binding interactions. Here, we present a method that identifies protein mutants with improved overall cellular efficacy, an objective not feasible with previous approaches. Mutated protein libraries were produced in soluble, active form by means of cell-free protein synthesis. The efficacy of each individual protein was determined at a uniform dosage with a high-throughput protein product assay followed by a cell-based functional assay without requiring protein purification. We validated our platform by first screening mock libraries of epidermal growth factor (EGF) for stimulation of cell proliferation. We then demonstrated its effectiveness by identifying EGF mutants with significantly enhanced mitogenic activity at low concentrations compared to that of wild-type EGF. This is the first report of EGF mutants with improved biological efficacy despite much previous effort. Our platform can be extended to engineer a broad range of proteins, offering a general method to evolve proteins for improved biological efficacy.
机译：定向进化是蛋白质工程的强大策略。然而，药物蛋白的进化受到当前筛选对结合相互作用的依赖的限制。在这里，我们提出了一种方法，该方法可识别具有改善的整体细胞效力的蛋白质突变体，而该方法在以前的方法中是不可行的。突变的蛋白质文库通过无细胞蛋白质合成以可溶性，活性形式产生。使用高通量蛋白质产物测定法，然后进行基于细胞的功能测定法，无需蛋白质纯化，即可在统一剂量下确定每种蛋白质的功效。我们通过首先筛选表皮生长因子（EGF）的模拟库来刺激细胞增殖来验证我们的平台。然后，我们通过鉴定与野生型EGF相比在低浓度下具有明显增强的有丝分裂活性的EGF突变体来证明其有效性。尽管有许多先前的努力，这是具有改善的生物功效的EGF突变体的首次报道。我们的平台可以扩展为工程化广泛的蛋白质，提供了进化蛋白质以提高生物学功效的通用方法。

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