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

doi:10.1093/protein/gzs033

CNU IR > Pharmacy and Science > Dept. of Pharmacy > Periodical Articles > Item 310902800/27620

Please use this identifier to cite or link to this item: https://ir.cnu.edu.tw/handle/310902800/27620

Title:	ECSTASY, an adjustable membrane-tethered/soluble protein expression system for the directed evolution of mammalian proteins
Authors:	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.
Contributors:	藥學系
Keywords:	Beta-Glucuronidase Directed Evolution Gpi Anchor High-Throughput Screening Mammalian Cell Surface Display
Date:	2012-07
Issue Date:	2014-03-21 16:15:30 (UTC+8)
Publisher:	Oxford Univ Press
Abstract:	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.
Relation:	Protein Engineering Design & Selection, 25(7), 367-375
Appears in Collections:	[Dept. of Pharmacy] Periodical Articles

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