Recombinant Protein Production in <i>E. coli</i> Using the phoA Expression System
Auto-inducible promoter systems have been reported to increase soluble product formation in the periplasm of <i>E. coli</i> compared to inducer-dependent systems. In this study, we investigated the phosphate (PO<sub>4</sub>)-sensitive phoA expression system (pAT) for the prod...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-04-01
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| Series: | Fermentation |
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| Online Access: | https://www.mdpi.com/2311-5637/8/4/181 |
| _version_ | 1797446491910963200 |
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| author | Thomas Gundinger Stefan Kittler Sabine Kubicek Julian Kopp Oliver Spadiut |
| author_facet | Thomas Gundinger Stefan Kittler Sabine Kubicek Julian Kopp Oliver Spadiut |
| author_sort | Thomas Gundinger |
| collection | DOAJ |
| description | Auto-inducible promoter systems have been reported to increase soluble product formation in the periplasm of <i>E. coli</i> compared to inducer-dependent systems. In this study, we investigated the phosphate (PO<sub>4</sub>)-sensitive phoA expression system (pAT) for the production of a recombinant model antigen-binding fragment (Fab) in the periplasm of <i>E. coli</i> in detail. We explored the impact of non-limiting and limiting PO<sub>4</sub> conditions on strain physiology as well as Fab productivity. We compared different methods for extracellular PO<sub>4</sub> detection, identifying automated colorimetric measurement to be most suitable for at-line PO<sub>4</sub> monitoring. We showed that PO<sub>4</sub> limitation boosts phoA-based gene expression, however, the product was already formed at non-limiting PO<sub>4</sub> conditions, indicating leaky expression. Furthermore, cultivation under PO<sub>4</sub> limitation caused physiological changes ultimately resulting in a metabolic breakdown at PO<sub>4</sub> starvation. Finally, we give recommendations for process optimization with the phoA expression system. In summary, our study provides very detailed information on the <i>E. coli</i> phoA expression system, thus extending the existing knowledge of this system, and underlines its high potential for the successful production of periplasmic products in <i>E. coli</i>. |
| first_indexed | 2024-03-09T13:41:09Z |
| format | Article |
| id | doaj.art-375faaa2ed994ec8b098374fada0458b |
| institution | Directory Open Access Journal |
| issn | 2311-5637 |
| language | English |
| last_indexed | 2024-03-09T13:41:09Z |
| publishDate | 2022-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Fermentation |
| spelling | doaj.art-375faaa2ed994ec8b098374fada0458b2023-11-30T21:06:21ZengMDPI AGFermentation2311-56372022-04-018418110.3390/fermentation8040181Recombinant Protein Production in <i>E. coli</i> Using the phoA Expression SystemThomas Gundinger0Stefan Kittler1Sabine Kubicek2Julian Kopp3Oliver Spadiut4Research Group Integrated Bioprocess Development, Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, Gumpendorferstraße 1a, 1060 Vienna, AustriaResearch Group Integrated Bioprocess Development, Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, Gumpendorferstraße 1a, 1060 Vienna, AustriaResearch Group Integrated Bioprocess Development, Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, Gumpendorferstraße 1a, 1060 Vienna, AustriaResearch Group Integrated Bioprocess Development, Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, Gumpendorferstraße 1a, 1060 Vienna, AustriaResearch Group Integrated Bioprocess Development, Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, Gumpendorferstraße 1a, 1060 Vienna, AustriaAuto-inducible promoter systems have been reported to increase soluble product formation in the periplasm of <i>E. coli</i> compared to inducer-dependent systems. In this study, we investigated the phosphate (PO<sub>4</sub>)-sensitive phoA expression system (pAT) for the production of a recombinant model antigen-binding fragment (Fab) in the periplasm of <i>E. coli</i> in detail. We explored the impact of non-limiting and limiting PO<sub>4</sub> conditions on strain physiology as well as Fab productivity. We compared different methods for extracellular PO<sub>4</sub> detection, identifying automated colorimetric measurement to be most suitable for at-line PO<sub>4</sub> monitoring. We showed that PO<sub>4</sub> limitation boosts phoA-based gene expression, however, the product was already formed at non-limiting PO<sub>4</sub> conditions, indicating leaky expression. Furthermore, cultivation under PO<sub>4</sub> limitation caused physiological changes ultimately resulting in a metabolic breakdown at PO<sub>4</sub> starvation. Finally, we give recommendations for process optimization with the phoA expression system. In summary, our study provides very detailed information on the <i>E. coli</i> phoA expression system, thus extending the existing knowledge of this system, and underlines its high potential for the successful production of periplasmic products in <i>E. coli</i>.https://www.mdpi.com/2311-5637/8/4/181<i>E. coli</i>phoA promoterT7lac promoterpATpETantibody fragment |
| spellingShingle | Thomas Gundinger Stefan Kittler Sabine Kubicek Julian Kopp Oliver Spadiut Recombinant Protein Production in <i>E. coli</i> Using the phoA Expression System Fermentation <i>E. coli</i> phoA promoter T7lac promoter pAT pET antibody fragment |
| title | Recombinant Protein Production in <i>E. coli</i> Using the phoA Expression System |
| title_full | Recombinant Protein Production in <i>E. coli</i> Using the phoA Expression System |
| title_fullStr | Recombinant Protein Production in <i>E. coli</i> Using the phoA Expression System |
| title_full_unstemmed | Recombinant Protein Production in <i>E. coli</i> Using the phoA Expression System |
| title_short | Recombinant Protein Production in <i>E. coli</i> Using the phoA Expression System |
| title_sort | recombinant protein production in i e coli i using the phoa expression system |
| topic | <i>E. coli</i> phoA promoter T7lac promoter pAT pET antibody fragment |
| url | https://www.mdpi.com/2311-5637/8/4/181 |
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