Chia Nan University of Pharmacy & Science Institutional Repository:Item 310902800/27620
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    標題: ECSTASY, an adjustable membrane-tethered/soluble protein expression system for the directed evolution of mammalian proteins
    作者: Chen, Cheng-Pao
    Hsieh, Yuan-Ting
    Prijovich, Zeljko M.
    Chuang, Huai-Yao
    Chen, Kai-Chuan
    Lu, Wei-Cheng
    Tseng, Qingzong
    Leu, Yu-Lin
    Cheng, Tian-Lu
    Roffler, Steve R.
    貢獻者: 藥學系
    關鍵字: Beta-Glucuronidase
    Directed Evolution
    Gpi Anchor
    High-Throughput Screening
    Mammalian Cell Surface Display
    日期: 2012-07
    上傳時間: 2014-03-21 16:15:30 (UTC+8)
    出版者: Oxford Univ Press
    摘要: We describe an adjustable membrane-tethered/soluble protein screening methodology termed ECSTASY (enzyme cleavable surface tethered all-purpose screening system) which combines the power of high-throughput fluorescence-activated cell sorting of membrane-tethered proteins with the flexibility of soluble assays for isolation of improved mammalian recombinant proteins. In this approach, retroviral transduction is employed to stably tether a library of protein variants on the surface of mammalian cells via a glycosyl phosphatidylinositol anchor. High-throughput fluorescence-activated cell sorting is used to array cells expressing properly folded and/or active protein variants on their surface into micro-titer culture plates. After culture to expand individual clones, treatment of cells with phosphatidylinositol-phospholipase C releases soluble protein variants for multiplex measurement of protein concentration, activity and/or function. We utilized ECSTASY to rapidly generate human beta-glucuronidase variants for cancer therapy by antibody-directed enzyme prodrug therapy with up to 30-fold greater potency to catalyze the hydrolysis of the clinically relevant camptothecin anti-cancer prodrug as compared with wild-type human beta-glucuronidase. A variety of recombinant proteins could be adjustably displayed on fibroblasts, suggesting that ECSTASY represents a general, simple and versatile methodology for high-throughput screening to accelerate sequence activity-based evolution of mammalian proteins.
    關聯: Protein Engineering Design & Selection, 25(7), 367-375
    显示于类别:[藥學系(所)] 期刊論文

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