A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.
Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of rea...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2016-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC5074584?pdf=render |
| _version_ | 1819086572998361088 |
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| author | Nitzan Krinsky Maya Kaduri Janna Shainsky-Roitman Mor Goldfeder Eran Ivanir Itai Benhar Yuval Shoham Avi Schroeder |
| author_facet | Nitzan Krinsky Maya Kaduri Janna Shainsky-Roitman Mor Goldfeder Eran Ivanir Itai Benhar Yuval Shoham Avi Schroeder |
| author_sort | Nitzan Krinsky |
| collection | DOAJ |
| description | Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40-150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins. |
| first_indexed | 2024-12-21T21:22:23Z |
| format | Article |
| id | doaj.art-530d3764cd534f0fa66f1de827966de7 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-21T21:22:23Z |
| publishDate | 2016-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-530d3764cd534f0fa66f1de827966de72022-12-21T18:49:51ZengPublic Library of Science (PLoS)PLoS ONE1932-62032016-01-011110e016513710.1371/journal.pone.0165137A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.Nitzan KrinskyMaya KaduriJanna Shainsky-RoitmanMor GoldfederEran IvanirItai BenharYuval ShohamAvi SchroederCell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40-150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.http://europepmc.org/articles/PMC5074584?pdf=render |
| spellingShingle | Nitzan Krinsky Maya Kaduri Janna Shainsky-Roitman Mor Goldfeder Eran Ivanir Itai Benhar Yuval Shoham Avi Schroeder A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis. PLoS ONE |
| title | A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis. |
| title_full | A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis. |
| title_fullStr | A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis. |
| title_full_unstemmed | A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis. |
| title_short | A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis. |
| title_sort | simple and rapid method for preparing a cell free bacterial lysate for protein synthesis |
| url | http://europepmc.org/articles/PMC5074584?pdf=render |
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