| Autor: |
Wei Gao, Ning Bu, Yuan Lu |
| Jazyk: |
angličtina |
| Rok vydání: |
2019 |
| Předmět: |
|
| Zdroj: |
Methods and Protocols, Vol 2, Iss 1, p 16 (2019) |
| Druh dokumentu: |
article |
| ISSN: |
2409-9279 |
| DOI: |
10.3390/mps2010016 |
| Popis: |
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. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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