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

Synthetic and systems Biotechnology 2019年第4期4卷 189-196页

作者： Yonatan Chemla Eden Ozer Michael Shaferman Ben Zaad Rambabu Dandela Lital Alfonta Department of Life Sciences and Ilse Katz Institute for Nanoscale Science and Technology Ben-Gurion University of the NegevP.O.Box.653Beer-Sheva8410501Israel Institute of Chemical Technology-Indian Oil Odisha campusIIT Kharagpur extension centerMouza SamantpuriBhubaneswar751013OdishaIndia

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.

关键词： cell free system Genetic code expansion Thio-lysine Simplified extract preparation

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