scholarly journals Using unnatural amino acids to selectively label proteins for cellular imaging: a cell biologist viewpoint

FEBS Journal ◽  
2020 ◽  
Author(s):  
Natalie Elia
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Cellular Imaging ◽  
Cell Biologist ◽  
A Cell

Translational incorporation of multiple unnatural amino acids in a cell-free protein synthesis system

2014 ◽  
Vol 19 (3) ◽  
pp. 426-432 ◽  
Author(s):  
Su-Jin Oh ◽  
Kyung-Ho Lee ◽  
Ho-Cheol Kim ◽  
Christy Catherine ◽  
Hyungdon Yun ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Unnatural Amino Acids ◽  
Synthesis System ◽  
Free Protein ◽  
A Cell ◽  
Protein Synthesis System ◽  
Cell Free Protein Synthesis

scholarly journals Simplified Methodology for a Modular and Genetically Expanded Protein Synthesis in Cell-Free Systems

2019 ◽  
Author(s):  
Yonatan Chemla ◽  
Eden Ozer ◽  
Michael Shaferman ◽  
Ben Zaad ◽  
Rambabu Dandela ◽  
...  
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
High Efficiency ◽  
Living Cells ◽  
Trna Synthetase ◽  
Complex Nature ◽  
Reporter Protein ◽  
A Cell ◽  
Genetic Code Expansion

ABSTRACTGenetic code expansion, which enables the site-specific incorporation of unnatural amino acids into proteins, has emerged as a new and powerful tool for protein engineering. Currently, it is mainly utilized inside living cells for a myriad of applications. However, utilization of this technology in a cell-free, reconstituted platform has several advantages over living systems. The common limitations to the employment of these systems are the laborious and complex nature of its preparation and utilization. Herein, 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 expansion. In addition, we propose and demonstrate a modular approach to its utilization. By 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 efficiency. We demonstrate this with the simultaneous incorporation of two different unnatural amino acids into a reporter protein. Finally, we demonstrate the advantage of cell-free systems over living cells for the incorporation of δ-thio-boc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNACUA and tRNA-synthetase pair, which can not be achieved in a living cell.

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