Cargando…
The optimization of in vitro high-throughput chemical lysis of Escherichia coli. Application to ACP domain of the polyketide synthase ppsC from Mycobacterium tuberculosis
Protein production in Escherichia coli involves high-level expression in a culture, followed by harvesting of the cells and finally their disruption, or lysis, to release the expressed proteins. We compare three high-throughput chemical lysis methods to sonication, using a robotic platform and metho...
Autores principales: | , , , , , |
---|---|
Formato: | Texto |
Lenguaje: | English |
Publicado: |
Springer Netherlands
2010
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855807/ https://www.ncbi.nlm.nih.gov/pubmed/20069378 http://dx.doi.org/10.1007/s10969-009-9077-8 |
_version_ | 1782180211597508608 |
---|---|
author | Listwan, Pawel Pédelacq, Jean-Denis Lockard, Meghan Bell, Carolyn Terwilliger, Thomas C. Waldo, Geoffrey S. |
author_facet | Listwan, Pawel Pédelacq, Jean-Denis Lockard, Meghan Bell, Carolyn Terwilliger, Thomas C. Waldo, Geoffrey S. |
author_sort | Listwan, Pawel |
collection | PubMed |
description | Protein production in Escherichia coli involves high-level expression in a culture, followed by harvesting of the cells and finally their disruption, or lysis, to release the expressed proteins. We compare three high-throughput chemical lysis methods to sonication, using a robotic platform and methodologies developed in our laboratory [1]. Under the same expression conditions, all lysis methods varied in the degree of released soluble proteins. With a set of 96 test proteins, we used our split GFP to quantify the soluble and insoluble protein fractions after lysis. Both the amount of soluble protein and the percentage recovered in the soluble fraction using SoluLyse® were well correlated with sonication. Two other methods, Bugbuster® and lysozyme, did not correlate well with sonication. Considering the effects of lysis methods on protein solubility is especially important when accurate protein solubility measurements are needed, for example, when testing adjuvants, growth media, temperature, or when establishing the effects of truncation or sequence variation on protein stability. |
format | Text |
id | pubmed-2855807 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | Springer Netherlands |
record_format | MEDLINE/PubMed |
spelling | pubmed-28558072010-04-20 The optimization of in vitro high-throughput chemical lysis of Escherichia coli. Application to ACP domain of the polyketide synthase ppsC from Mycobacterium tuberculosis Listwan, Pawel Pédelacq, Jean-Denis Lockard, Meghan Bell, Carolyn Terwilliger, Thomas C. Waldo, Geoffrey S. J Struct Funct Genomics Article Protein production in Escherichia coli involves high-level expression in a culture, followed by harvesting of the cells and finally their disruption, or lysis, to release the expressed proteins. We compare three high-throughput chemical lysis methods to sonication, using a robotic platform and methodologies developed in our laboratory [1]. Under the same expression conditions, all lysis methods varied in the degree of released soluble proteins. With a set of 96 test proteins, we used our split GFP to quantify the soluble and insoluble protein fractions after lysis. Both the amount of soluble protein and the percentage recovered in the soluble fraction using SoluLyse® were well correlated with sonication. Two other methods, Bugbuster® and lysozyme, did not correlate well with sonication. Considering the effects of lysis methods on protein solubility is especially important when accurate protein solubility measurements are needed, for example, when testing adjuvants, growth media, temperature, or when establishing the effects of truncation or sequence variation on protein stability. Springer Netherlands 2010-01-13 2010 /pmc/articles/PMC2855807/ /pubmed/20069378 http://dx.doi.org/10.1007/s10969-009-9077-8 Text en © The Author(s) 2010 https://creativecommons.org/licenses/by-nc/4.0/ This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. |
spellingShingle | Article Listwan, Pawel Pédelacq, Jean-Denis Lockard, Meghan Bell, Carolyn Terwilliger, Thomas C. Waldo, Geoffrey S. The optimization of in vitro high-throughput chemical lysis of Escherichia coli. Application to ACP domain of the polyketide synthase ppsC from Mycobacterium tuberculosis |
title | The optimization of in vitro high-throughput chemical lysis of Escherichia coli. Application to ACP domain of the polyketide synthase ppsC from Mycobacterium tuberculosis |
title_full | The optimization of in vitro high-throughput chemical lysis of Escherichia coli. Application to ACP domain of the polyketide synthase ppsC from Mycobacterium tuberculosis |
title_fullStr | The optimization of in vitro high-throughput chemical lysis of Escherichia coli. Application to ACP domain of the polyketide synthase ppsC from Mycobacterium tuberculosis |
title_full_unstemmed | The optimization of in vitro high-throughput chemical lysis of Escherichia coli. Application to ACP domain of the polyketide synthase ppsC from Mycobacterium tuberculosis |
title_short | The optimization of in vitro high-throughput chemical lysis of Escherichia coli. Application to ACP domain of the polyketide synthase ppsC from Mycobacterium tuberculosis |
title_sort | optimization of in vitro high-throughput chemical lysis of escherichia coli. application to acp domain of the polyketide synthase ppsc from mycobacterium tuberculosis |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855807/ https://www.ncbi.nlm.nih.gov/pubmed/20069378 http://dx.doi.org/10.1007/s10969-009-9077-8 |
work_keys_str_mv | AT listwanpawel theoptimizationofinvitrohighthroughputchemicallysisofescherichiacoliapplicationtoacpdomainofthepolyketidesynthaseppscfrommycobacteriumtuberculosis AT pedelacqjeandenis theoptimizationofinvitrohighthroughputchemicallysisofescherichiacoliapplicationtoacpdomainofthepolyketidesynthaseppscfrommycobacteriumtuberculosis AT lockardmeghan theoptimizationofinvitrohighthroughputchemicallysisofescherichiacoliapplicationtoacpdomainofthepolyketidesynthaseppscfrommycobacteriumtuberculosis AT bellcarolyn theoptimizationofinvitrohighthroughputchemicallysisofescherichiacoliapplicationtoacpdomainofthepolyketidesynthaseppscfrommycobacteriumtuberculosis AT terwilligerthomasc theoptimizationofinvitrohighthroughputchemicallysisofescherichiacoliapplicationtoacpdomainofthepolyketidesynthaseppscfrommycobacteriumtuberculosis AT waldogeoffreys theoptimizationofinvitrohighthroughputchemicallysisofescherichiacoliapplicationtoacpdomainofthepolyketidesynthaseppscfrommycobacteriumtuberculosis AT listwanpawel optimizationofinvitrohighthroughputchemicallysisofescherichiacoliapplicationtoacpdomainofthepolyketidesynthaseppscfrommycobacteriumtuberculosis AT pedelacqjeandenis optimizationofinvitrohighthroughputchemicallysisofescherichiacoliapplicationtoacpdomainofthepolyketidesynthaseppscfrommycobacteriumtuberculosis AT lockardmeghan optimizationofinvitrohighthroughputchemicallysisofescherichiacoliapplicationtoacpdomainofthepolyketidesynthaseppscfrommycobacteriumtuberculosis AT bellcarolyn optimizationofinvitrohighthroughputchemicallysisofescherichiacoliapplicationtoacpdomainofthepolyketidesynthaseppscfrommycobacteriumtuberculosis AT terwilligerthomasc optimizationofinvitrohighthroughputchemicallysisofescherichiacoliapplicationtoacpdomainofthepolyketidesynthaseppscfrommycobacteriumtuberculosis AT waldogeoffreys optimizationofinvitrohighthroughputchemicallysisofescherichiacoliapplicationtoacpdomainofthepolyketidesynthaseppscfrommycobacteriumtuberculosis |