Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems

作     者：Yonatan Chemla Eden Ozer Michael Shaferman Ben Zaad Rambabu Dandela Lital Alfonta 

作者机构：Department of Life Sciences and Ilse Katz Institute for Nanoscale Science and TechnologyBen-Gurion University of the NegevP.O.Box.653Beer-Sheva8410501Israel Institute of Chemical Technology-Indian OilOdisha campusIIT Kharagpur extension centerMouza SamantpuriBhubaneswar751013OdishaIndia 

出 版 物：《Synthetic and Systems Biotechnology》 (合成和系统生物技术（英文）)

年 卷 期：2019年第4卷第4期

页      面：189-196页

核心收录：

学科分类：0831[工学-生物医学工程（可授工学、理学、医学学位）] 0710[理学-生物学] 081704[工学-应用化学] 1001[医学-基础医学(可授医学、理学学位)] 07[理学] 08[工学] 0817[工学-化学工程与技术] 070303[理学-有机化学] 0703[理学-化学] 0836[工学-生物工程] 

基　　金：Seventh Framework Programme, FP7, (260647) European Research Council, ERC 

主　　题：Cell free system Genetic code expansion Thio-lysine Simplified extract preparation 

摘      要：Genetic code expansion,which enables the site-specific incorporation of unnatural amino acids into proteins,has emerged as a new and powerful tool for protein ***,it is mainly utilized inside living cells for a myriad of ***,the utilization of this technology in a cell-free,reconstituted platform has several advantages over living *** typical limitations to the employment of these systems are the laborious and complex nature of its preparation and ***,we describe a simplified method for the preparation of this system from Escherichia coli cells,which is specifically adapted for the expression of the components needed for cell-free genetic code ***,we propose and demonstrate a modular approach to its *** this approach,it is possible to prepare and store different extracts,harboring various translational components,and mix and match them as needed for more than four years retaining its high *** demonstrate this with the simultaneous incorporation of two different unnatural amino acids into a reporter ***,we demonstrate the advantage of cell-free systems over living cells for the incorporation ofδ-thio-boc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNACUA and tRNA-synthetase pair,which could not be achieved in a living cell.