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利用强大的无细胞系统将非天然氨基酸高效掺入蛋白质中。

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

作者信息

Gao Wei, Bu Ning, Lu Yuan

机构信息

College of Life Science, Shenyang Normal University, Shenyang 100034, Liaoning, China.

Department of Chemical Engineering, Tsinghua University, Beijing 100084, China.

出版信息

Methods Protoc. 2019 Feb 12;2(1):16. doi: 10.3390/mps2010016.

DOI:10.3390/mps2010016
PMID:31164598
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6481062/
Abstract

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 crude extract, with unnatural orthogonal aminoacyl-tRNA synthetase and suppressor tRNA evolved from . The superfolder green fluorescent protein (sfGFP) and -propargyloxyphenylalanine (PaF) 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.

摘要

非天然蛋白质是至关重要的生物大分子，已广泛应用于基础科学、新型生物聚合物材料、酶和治疗学领域。无细胞蛋白质合成（CFPS）系统可作为一个强大的平台，通过高效位点特异性掺入非天然氨基酸（UNAAs）来合成非天然蛋白质，不受细胞膜通透性和非天然成分毒性的限制。在此，我们描述了一种基于粗提物在CFPS系统中合成非天然蛋白质的快速简便方法，该方法使用了从[具体来源]进化而来的非天然正交氨酰-tRNA合成酶和抑制性tRNA。超折叠绿色荧光蛋白（sfGFP）和对炔丙氧基苯丙氨酸（PaF）用作模型蛋白和非天然氨基酸。通过微孔板分光光度计、亲和色谱和液相色谱-质谱/质谱（LC-MS/MS）对非天然sfGFP的合成进行了表征。本方案提供了一个详细的程序，指导如何使用强大的CFPS系统按需合成非天然蛋白质。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4374/6481062/3365b4f09c6a/mps-02-00016-g001.jpg

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本文引用的文献

1
Cell-free synthetic biology: Engineering in an open world.
Synth Syst Biotechnol. 2017 Mar 3;2(1):23-27. doi: 10.1016/j.synbio.2017.02.003. eCollection 2017 Mar.
2
Translation system engineering in Escherichia coli enhances non-canonical amino acid incorporation into proteins.
Biotechnol Bioeng. 2017 May;114(5):1074-1086. doi: 10.1002/bit.26239. Epub 2017 Feb 2.
3
Genetically encoding new bioreactivity.
N Biotechnol. 2017 Sep 25;38(Pt A):16-25. doi: 10.1016/j.nbt.2016.10.003. Epub 2016 Oct 6.
4
Incorporation of Non-Canonical Amino Acids.
Adv Exp Med Biol. 2015;869:119-51. doi: 10.1007/978-1-4939-2845-3_7.
5
Robust production of recombinant phosphoproteins using cell-free protein synthesis.
Nat Commun. 2015 Sep 9;6:8168. doi: 10.1038/ncomms9168.
6
Repurposing the translation apparatus for synthetic biology.
Curr Opin Chem Biol. 2015 Oct;28:83-90. doi: 10.1016/j.cbpa.2015.06.008. Epub 2015 Jul 15.
7
Cotranslational incorporation of non-standard amino acids using cell-free protein synthesis.
FEBS Lett. 2015 Jul 8;589(15):1703-12. doi: 10.1016/j.febslet.2015.04.041. Epub 2015 May 1.
8
Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis.
Front Chem. 2014 Jun 10;2:34. doi: 10.3389/fchem.2014.00034. eCollection 2014.
9
Recoding the genetic code with selenocysteine.
Angew Chem Int Ed Engl. 2014 Jan 3;53(1):319-23. doi: 10.1002/anie.201308584.
10
Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system.
Biochimie. 2014 Apr;99:162-8. doi: 10.1016/j.biochi.2013.11.025. Epub 2013 Dec 8.

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