A Competitive Flow Cytometry Screening System for Directed Evolution of Therapeutic Enzyme

详细信息    查看全文

• 作者：Feng Cheng ; Tsvetan Kardashliev ; Christian Pitzler ; Aamir Shehzad ; Hongqi Lue ; J眉rgen Bernhagen ; Leilei Zhu ; Ulrich Schwaneberg
• 刊名：ACS Synthetic Biology
• 出版年：2015
• 出版时间：July 17, 2015
• 年：2015
• 卷：4
• 期：7
• 页码：768-775
• 全文大小：417K
• ISSN：2161-5063

文摘

A ligand-mediated eGFP-expression system (LiMEx) was developed as a novel flow cytometry based screening platform that relies on a competitive conversion/binding of arginine between arginine deiminase and arginine repressor. Unlike product-driven detection systems, the competitive screening platform allows to evolve enzymes toward efficient operation at low substrate concentrations under physiological conditions. The principle of LiMEx was validated by evolving arginine deiminase (ADI, an anticancer therapeutic) for stronger inhibition of tumor growth. After screening of 鈭?.2 脳 10⁶ clones in three iterative rounds of epPCR libraries, PpADI (ADI from Pseudomonas plecoglossicida) variant M31 with reduced S_0.5 value (0.17 mM compared to 1.23 mM (WT)) and, importantly, increased activity at physiological arginine concentration (M31:6.14 s^鈥?; WT: not detectable) was identified. Moreover, M31 showed a significant inhibitory effect against SK-MEL-28 and G361 melanoma cell lines. (IC₅₀ = 0.02 渭g/mL for SK-MEL-28 and G361).