A scalable screening of E. coli strains for recombinant protein expression
Structural biology projects are highly dependent on the large-scale expression of soluble protein and, for this purpose, heterologous expression using bacteria or yeast as host systems is usually employed. In this scenario, some of the parameters to be optimized include ( i ) those related to the pr...
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crplos:10.1371/journal.pone.0271403 2024-06-23T07:50:16+00:00 A scalable screening of E. coli strains for recombinant protein expression Morão, Luana G. Manzine, Lívia R. Clementino, Lívia Oliveira D. Wrenger, Carsten Nascimento, Alessandro S. Beddoe, Travis Fundação de Amparo à Pesquisa do Estado de São Paulo Fundação de Amparo à Pesquisa do Estado de São Paulo Fundação de Amparo à Pesquisa do Estado de São Paulo Fundação de Amparo à Pesquisa do Estado de São Paulo Fundação de Amparo à Pesquisa do Estado de São Paulo Conselho Nacional de Desenvolvimento Científico e Tecnológico Conselho Nacional de Desenvolvimento Científico e Tecnológico Conselho Nacional de Desenvolvimento Científico e Tecnológico 2022 http://dx.doi.org/10.1371/journal.pone.0271403 https://dx.plos.org/10.1371/journal.pone.0271403 en eng Public Library of Science (PLoS) http://creativecommons.org/licenses/by/4.0/ PLOS ONE volume 17, issue 7, page e0271403 ISSN 1932-6203 journal-article 2022 crplos https://doi.org/10.1371/journal.pone.0271403 2024-06-04T06:20:15Z Structural biology projects are highly dependent on the large-scale expression of soluble protein and, for this purpose, heterologous expression using bacteria or yeast as host systems is usually employed. In this scenario, some of the parameters to be optimized include ( i ) those related to the protein construct, such as the use of a fusion protein, the choice of an N-terminus fusion/tag or a C-terminus fusion/tag; ( ii ) those related to the expression stage, such as the concentration and selection of inducer agent and temperature expression and ( iii ) the choice of the host system, which includes the selection of a prokaryotic or eukaryotic cell and the adoption of a strain. The optimization of some of the parameters related to protein expression, stage ( ii ), is straightforward. On the other hand, the determination of the most suitable parameters related to protein construction requires a new cycle of gene cloning, while the optimization of the host cell is less straightforward. Here, we evaluated a scalable approach for the screening of host cells for protein expression in a structural biology pipeline. We evaluated four Escherichia coli strains looking for the best yield of soluble heterologous protein expression using the same strategy for protein construction and gene cloning and comparing it to our standard strain, Rosetta 2 (DE3). Using a liquid handling device (robot), E . coli pT-GroE, Lemo21(DE3), Arctic Express (DE3), and Rosetta Gami 2 (DE3) strains were screened for the maximal yield of soluble heterologous protein recovery. For the genes used in this experiment, the Arctic Express (DE3) strain resulted in better yields of soluble heterologous proteins. We propose that screening of host cell/strain is feasible, even for smaller laboratories and the experiment as proposed can easily be scalable to a high-throughput approach. Article in Journal/Newspaper Arctic PLOS Arctic PLOS ONE 17 7 e0271403 |
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description |
Structural biology projects are highly dependent on the large-scale expression of soluble protein and, for this purpose, heterologous expression using bacteria or yeast as host systems is usually employed. In this scenario, some of the parameters to be optimized include ( i ) those related to the protein construct, such as the use of a fusion protein, the choice of an N-terminus fusion/tag or a C-terminus fusion/tag; ( ii ) those related to the expression stage, such as the concentration and selection of inducer agent and temperature expression and ( iii ) the choice of the host system, which includes the selection of a prokaryotic or eukaryotic cell and the adoption of a strain. The optimization of some of the parameters related to protein expression, stage ( ii ), is straightforward. On the other hand, the determination of the most suitable parameters related to protein construction requires a new cycle of gene cloning, while the optimization of the host cell is less straightforward. Here, we evaluated a scalable approach for the screening of host cells for protein expression in a structural biology pipeline. We evaluated four Escherichia coli strains looking for the best yield of soluble heterologous protein expression using the same strategy for protein construction and gene cloning and comparing it to our standard strain, Rosetta 2 (DE3). Using a liquid handling device (robot), E . coli pT-GroE, Lemo21(DE3), Arctic Express (DE3), and Rosetta Gami 2 (DE3) strains were screened for the maximal yield of soluble heterologous protein recovery. For the genes used in this experiment, the Arctic Express (DE3) strain resulted in better yields of soluble heterologous proteins. We propose that screening of host cell/strain is feasible, even for smaller laboratories and the experiment as proposed can easily be scalable to a high-throughput approach. |
author2 |
Beddoe, Travis Fundação de Amparo à Pesquisa do Estado de São Paulo Fundação de Amparo à Pesquisa do Estado de São Paulo Fundação de Amparo à Pesquisa do Estado de São Paulo Fundação de Amparo à Pesquisa do Estado de São Paulo Fundação de Amparo à Pesquisa do Estado de São Paulo Conselho Nacional de Desenvolvimento Científico e Tecnológico Conselho Nacional de Desenvolvimento Científico e Tecnológico Conselho Nacional de Desenvolvimento Científico e Tecnológico |
format |
Article in Journal/Newspaper |
author |
Morão, Luana G. Manzine, Lívia R. Clementino, Lívia Oliveira D. Wrenger, Carsten Nascimento, Alessandro S. |
spellingShingle |
Morão, Luana G. Manzine, Lívia R. Clementino, Lívia Oliveira D. Wrenger, Carsten Nascimento, Alessandro S. A scalable screening of E. coli strains for recombinant protein expression |
author_facet |
Morão, Luana G. Manzine, Lívia R. Clementino, Lívia Oliveira D. Wrenger, Carsten Nascimento, Alessandro S. |
author_sort |
Morão, Luana G. |
title |
A scalable screening of E. coli strains for recombinant protein expression |
title_short |
A scalable screening of E. coli strains for recombinant protein expression |
title_full |
A scalable screening of E. coli strains for recombinant protein expression |
title_fullStr |
A scalable screening of E. coli strains for recombinant protein expression |
title_full_unstemmed |
A scalable screening of E. coli strains for recombinant protein expression |
title_sort |
scalable screening of e. coli strains for recombinant protein expression |
publisher |
Public Library of Science (PLoS) |
publishDate |
2022 |
url |
http://dx.doi.org/10.1371/journal.pone.0271403 https://dx.plos.org/10.1371/journal.pone.0271403 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLOS ONE volume 17, issue 7, page e0271403 ISSN 1932-6203 |
op_rights |
http://creativecommons.org/licenses/by/4.0/ |
op_doi |
https://doi.org/10.1371/journal.pone.0271403 |
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PLOS ONE |
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17 |
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7 |
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e0271403 |
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