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Efficient Incorporation of Unnatural Amino Acids into Proteins with a Robust Cell-Free System

by Wei Gao 1,2, Ning Bu 1,* and Yuan Lu 2,3,4,*
1
College of Life Science, Shenyang Normal University, Shenyang 100034, Liaoning, China
2
Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
3
Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
4
Key Lab of Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
*
Authors to whom correspondence should be addressed.
Methods Protoc. 2019, 2(1), 16; https://doi.org/10.3390/mps2010016
Received: 16 January 2019 / Revised: 7 February 2019 / Accepted: 7 February 2019 / Published: 12 February 2019
(This article belongs to the Special Issue Cell-Free Synthetic Biology)
Unnatural proteins are crucial biomacromolecules and have been widely applied in fundamental science, novel biopolymer materials, enzymes, and therapeutics. Cell-free protein synthesis (CFPS) system can serve as a robust platform to synthesize unnatural proteins by highly effective site-specific incorporation of unnatural amino acids (UNAAs), without the limitations of cell membrane permeability and the toxicity of unnatural components. Here, we describe a quick and simple method to synthesize unnatural proteins in CFPS system based on Escherichia coli crude extract, with unnatural orthogonal aminoacyl-tRNA synthetase and suppressor tRNA evolved from Methanocaldococcus jannaschii. The superfolder green fluorescent protein (sfGFP) and p-propargyloxyphenylalanine (pPaF) were used as the model protein and UNAA. The synthesis of unnatural sfGFPs was characterized by microplate spectrophotometer, affinity chromatography, and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). This protocol provides a detailed procedure guiding how to use the powerful CFPS system to synthesize unnatural proteins on demand. View Full-Text
Keywords: cell-free protein synthesis; unnatural amino acid; unnatural protein cell-free protein synthesis; unnatural amino acid; unnatural protein
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Gao, W.; Bu, N.; Lu, Y. Efficient Incorporation of Unnatural Amino Acids into Proteins with a Robust Cell-Free System. Methods Protoc. 2019, 2, 16.

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