ECSTASY, an adjustable membrane-tethered/soluble protein expression system for the directed evolution of mammalian proteins

doi:10.1093/protein/gzs033

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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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