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Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption
Cell-free protein synthesis (CFPS) is a powerful tool for the preparation of toxic proteins, directed protein evolution, and bottom-up synthetic biology. The transcription-translation machinery for CFPS is provided by cell extracts, which usually contain 20–30 mg/mL of proteins. In general, these ce...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4851396/ https://www.ncbi.nlm.nih.gov/pubmed/27128597 http://dx.doi.org/10.1371/journal.pone.0154614 |
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author | Fujiwara, Kei Doi, Nobuhide |
author_facet | Fujiwara, Kei Doi, Nobuhide |
author_sort | Fujiwara, Kei |
collection | PubMed |
description | Cell-free protein synthesis (CFPS) is a powerful tool for the preparation of toxic proteins, directed protein evolution, and bottom-up synthetic biology. The transcription-translation machinery for CFPS is provided by cell extracts, which usually contain 20–30 mg/mL of proteins. In general, these cell extracts are prepared by physical disruption; however, this requires technical experience and special machinery. Here, we report a method to prepare cell extracts for CFPS using a biochemical method, which disrupts cells through the combination of lysozyme treatment, osmotic shock, and freeze-thaw cycles. The resulting cell extracts showed similar features to those obtained by physical disruption, and was able to synthesize active green fluorescent proteins in the presence of appropriate chemicals to a concentration of 20 μM (0.5 mg/mL). |
format | Online Article Text |
id | pubmed-4851396 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-48513962016-05-07 Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption Fujiwara, Kei Doi, Nobuhide PLoS One Research Article Cell-free protein synthesis (CFPS) is a powerful tool for the preparation of toxic proteins, directed protein evolution, and bottom-up synthetic biology. The transcription-translation machinery for CFPS is provided by cell extracts, which usually contain 20–30 mg/mL of proteins. In general, these cell extracts are prepared by physical disruption; however, this requires technical experience and special machinery. Here, we report a method to prepare cell extracts for CFPS using a biochemical method, which disrupts cells through the combination of lysozyme treatment, osmotic shock, and freeze-thaw cycles. The resulting cell extracts showed similar features to those obtained by physical disruption, and was able to synthesize active green fluorescent proteins in the presence of appropriate chemicals to a concentration of 20 μM (0.5 mg/mL). Public Library of Science 2016-04-29 /pmc/articles/PMC4851396/ /pubmed/27128597 http://dx.doi.org/10.1371/journal.pone.0154614 Text en © 2016 Fujiwara, Doi http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Fujiwara, Kei Doi, Nobuhide Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption |
title | Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption |
title_full | Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption |
title_fullStr | Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption |
title_full_unstemmed | Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption |
title_short | Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption |
title_sort | biochemical preparation of cell extract for cell-free protein synthesis without physical disruption |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4851396/ https://www.ncbi.nlm.nih.gov/pubmed/27128597 http://dx.doi.org/10.1371/journal.pone.0154614 |
work_keys_str_mv | AT fujiwarakei biochemicalpreparationofcellextractforcellfreeproteinsynthesiswithoutphysicaldisruption AT doinobuhide biochemicalpreparationofcellextractforcellfreeproteinsynthesiswithoutphysicaldisruption |