ECSTASY, an adjustable membrane-tethered/soluble protein expression system for the directed evolution of mammalian proteins.
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 microtiter 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 β-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 β-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.
Institute of Biomedical Sciences, Academia Sinica, Taipei 11529,  Taiwan.
Cheng-Pao
CP
Chen
Yuan-Ting
YT
Hsieh
Zeljko M
ZM
Prijovich
Huai-Yao
HY
Chuang
Kai-Chuan
KC
Chen
Wei-Cheng
WC
Lu
Qingzong
Q
Tseng
Yu-Lin
YL
Leu
Tian-Lu
TL
Cheng
Steve R
SR
Roffler
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D058851:GPI-Linked Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D051379:Mice; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D058851:GPI-Linked Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D005966:Glucuronidase