Detailed small-scale characterization and scale-up of active YFP inclusion body production with Escherichia coli induced by a tetrameric coiled coil domain
During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been de...
Ausführliche Beschreibung
Gespeichert in:
| Autor*in: |
Lamm, Robin [verfasserIn] |
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| Format: |
E-Artikel |
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| Sprache: |
Englisch |
| Erschienen: |
2020transfer abstract |
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| Schlagwörter: |
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| Umfang: |
11 |
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| Übergeordnetes Werk: |
Enthalten in: Oral Squamous Cell Carcinoma in Three Related Kowari (Dasyuroides byrnei) - Saunders, Richard ELSEVIER, 2017, Amsterdam [u.a.] |
|---|---|
| Übergeordnetes Werk: |
volume:129 ; year:2020 ; number:6 ; pages:730-740 ; extent:11 |
| Links: |
|---|
| DOI / URN: |
10.1016/j.jbiosc.2020.02.003 |
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ELV050062808 |
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| 245 | 1 | 0 | |a Detailed small-scale characterization and scale-up of active YFP inclusion body production with Escherichia coli induced by a tetrameric coiled coil domain |
| 264 | 1 | |c 2020transfer abstract | |
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| 520 | |a During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37 °C to 15 °C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12 g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass. | ||
| 520 | |a During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37 °C to 15 °C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12 g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass. | ||
| 650 | 7 | |a Fluorescent protein |2 Elsevier | |
| 650 | 7 | |a Process characterization |2 Elsevier | |
| 650 | 7 | |a High-throughput technologies |2 Elsevier | |
| 650 | 7 | |a Online monitoring |2 Elsevier | |
| 650 | 7 | |a Active inclusion bodies |2 Elsevier | |
| 650 | 7 | |a Scale-up |2 Elsevier | |
| 700 | 1 | |a Jäger, Vera D. |4 oth | |
| 700 | 1 | |a Heyman, Benedikt |4 oth | |
| 700 | 1 | |a Berg, Christoph |4 oth | |
| 700 | 1 | |a Cürten, Christin |4 oth | |
| 700 | 1 | |a Krauss, Ulrich |4 oth | |
| 700 | 1 | |a Jaeger, Karl-Erich |4 oth | |
| 700 | 1 | |a Büchs, Jochen |4 oth | |
| 773 | 0 | 8 | |i Enthalten in |n Elsevier Science |a Saunders, Richard ELSEVIER |t Oral Squamous Cell Carcinoma in Three Related Kowari (Dasyuroides byrnei) |d 2017 |g Amsterdam [u.a.] |w (DE-627)ELV020602480 |
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10.1016/j.jbiosc.2020.02.003 doi /cbs_pica/cbs_olc/import_discovery/elsevier/einzuspielen/GBV00000000000983.pica (DE-627)ELV050062808 (ELSEVIER)S1389-1723(19)31010-2 DE-627 ger DE-627 rakwb eng 610 VZ 540 660 VZ 58.34 bkl Lamm, Robin verfasserin aut Detailed small-scale characterization and scale-up of active YFP inclusion body production with Escherichia coli induced by a tetrameric coiled coil domain 2020transfer abstract 11 nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37 °C to 15 °C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12 g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass. During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37 °C to 15 °C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12 g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass. Fluorescent protein Elsevier Process characterization Elsevier High-throughput technologies Elsevier Online monitoring Elsevier Active inclusion bodies Elsevier Scale-up Elsevier Jäger, Vera D. oth Heyman, Benedikt oth Berg, Christoph oth Cürten, Christin oth Krauss, Ulrich oth Jaeger, Karl-Erich oth Büchs, Jochen oth Enthalten in Elsevier Science Saunders, Richard ELSEVIER Oral Squamous Cell Carcinoma in Three Related Kowari (Dasyuroides byrnei) 2017 Amsterdam [u.a.] (DE-627)ELV020602480 volume:129 year:2020 number:6 pages:730-740 extent:11 https://doi.org/10.1016/j.jbiosc.2020.02.003 Volltext GBV_USEFLAG_U GBV_ELV SYSFLAG_U SSG-OLC-PHA 58.34 Lebensmitteltechnologie VZ AR 129 2020 6 730-740 11 |
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10.1016/j.jbiosc.2020.02.003 doi /cbs_pica/cbs_olc/import_discovery/elsevier/einzuspielen/GBV00000000000983.pica (DE-627)ELV050062808 (ELSEVIER)S1389-1723(19)31010-2 DE-627 ger DE-627 rakwb eng 610 VZ 540 660 VZ 58.34 bkl Lamm, Robin verfasserin aut Detailed small-scale characterization and scale-up of active YFP inclusion body production with Escherichia coli induced by a tetrameric coiled coil domain 2020transfer abstract 11 nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37 °C to 15 °C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12 g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass. During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37 °C to 15 °C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12 g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass. Fluorescent protein Elsevier Process characterization Elsevier High-throughput technologies Elsevier Online monitoring Elsevier Active inclusion bodies Elsevier Scale-up Elsevier Jäger, Vera D. oth Heyman, Benedikt oth Berg, Christoph oth Cürten, Christin oth Krauss, Ulrich oth Jaeger, Karl-Erich oth Büchs, Jochen oth Enthalten in Elsevier Science Saunders, Richard ELSEVIER Oral Squamous Cell Carcinoma in Three Related Kowari (Dasyuroides byrnei) 2017 Amsterdam [u.a.] (DE-627)ELV020602480 volume:129 year:2020 number:6 pages:730-740 extent:11 https://doi.org/10.1016/j.jbiosc.2020.02.003 Volltext GBV_USEFLAG_U GBV_ELV SYSFLAG_U SSG-OLC-PHA 58.34 Lebensmitteltechnologie VZ AR 129 2020 6 730-740 11 |
| allfields_unstemmed |
10.1016/j.jbiosc.2020.02.003 doi /cbs_pica/cbs_olc/import_discovery/elsevier/einzuspielen/GBV00000000000983.pica (DE-627)ELV050062808 (ELSEVIER)S1389-1723(19)31010-2 DE-627 ger DE-627 rakwb eng 610 VZ 540 660 VZ 58.34 bkl Lamm, Robin verfasserin aut Detailed small-scale characterization and scale-up of active YFP inclusion body production with Escherichia coli induced by a tetrameric coiled coil domain 2020transfer abstract 11 nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37 °C to 15 °C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12 g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass. During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37 °C to 15 °C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12 g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass. Fluorescent protein Elsevier Process characterization Elsevier High-throughput technologies Elsevier Online monitoring Elsevier Active inclusion bodies Elsevier Scale-up Elsevier Jäger, Vera D. oth Heyman, Benedikt oth Berg, Christoph oth Cürten, Christin oth Krauss, Ulrich oth Jaeger, Karl-Erich oth Büchs, Jochen oth Enthalten in Elsevier Science Saunders, Richard ELSEVIER Oral Squamous Cell Carcinoma in Three Related Kowari (Dasyuroides byrnei) 2017 Amsterdam [u.a.] (DE-627)ELV020602480 volume:129 year:2020 number:6 pages:730-740 extent:11 https://doi.org/10.1016/j.jbiosc.2020.02.003 Volltext GBV_USEFLAG_U GBV_ELV SYSFLAG_U SSG-OLC-PHA 58.34 Lebensmitteltechnologie VZ AR 129 2020 6 730-740 11 |
| allfieldsGer |
10.1016/j.jbiosc.2020.02.003 doi /cbs_pica/cbs_olc/import_discovery/elsevier/einzuspielen/GBV00000000000983.pica (DE-627)ELV050062808 (ELSEVIER)S1389-1723(19)31010-2 DE-627 ger DE-627 rakwb eng 610 VZ 540 660 VZ 58.34 bkl Lamm, Robin verfasserin aut Detailed small-scale characterization and scale-up of active YFP inclusion body production with Escherichia coli induced by a tetrameric coiled coil domain 2020transfer abstract 11 nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37 °C to 15 °C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12 g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass. During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37 °C to 15 °C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12 g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass. Fluorescent protein Elsevier Process characterization Elsevier High-throughput technologies Elsevier Online monitoring Elsevier Active inclusion bodies Elsevier Scale-up Elsevier Jäger, Vera D. oth Heyman, Benedikt oth Berg, Christoph oth Cürten, Christin oth Krauss, Ulrich oth Jaeger, Karl-Erich oth Büchs, Jochen oth Enthalten in Elsevier Science Saunders, Richard ELSEVIER Oral Squamous Cell Carcinoma in Three Related Kowari (Dasyuroides byrnei) 2017 Amsterdam [u.a.] (DE-627)ELV020602480 volume:129 year:2020 number:6 pages:730-740 extent:11 https://doi.org/10.1016/j.jbiosc.2020.02.003 Volltext GBV_USEFLAG_U GBV_ELV SYSFLAG_U SSG-OLC-PHA 58.34 Lebensmitteltechnologie VZ AR 129 2020 6 730-740 11 |
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10.1016/j.jbiosc.2020.02.003 doi /cbs_pica/cbs_olc/import_discovery/elsevier/einzuspielen/GBV00000000000983.pica (DE-627)ELV050062808 (ELSEVIER)S1389-1723(19)31010-2 DE-627 ger DE-627 rakwb eng 610 VZ 540 660 VZ 58.34 bkl Lamm, Robin verfasserin aut Detailed small-scale characterization and scale-up of active YFP inclusion body production with Escherichia coli induced by a tetrameric coiled coil domain 2020transfer abstract 11 nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37 °C to 15 °C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12 g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass. During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37 °C to 15 °C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12 g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass. Fluorescent protein Elsevier Process characterization Elsevier High-throughput technologies Elsevier Online monitoring Elsevier Active inclusion bodies Elsevier Scale-up Elsevier Jäger, Vera D. oth Heyman, Benedikt oth Berg, Christoph oth Cürten, Christin oth Krauss, Ulrich oth Jaeger, Karl-Erich oth Büchs, Jochen oth Enthalten in Elsevier Science Saunders, Richard ELSEVIER Oral Squamous Cell Carcinoma in Three Related Kowari (Dasyuroides byrnei) 2017 Amsterdam [u.a.] (DE-627)ELV020602480 volume:129 year:2020 number:6 pages:730-740 extent:11 https://doi.org/10.1016/j.jbiosc.2020.02.003 Volltext GBV_USEFLAG_U GBV_ELV SYSFLAG_U SSG-OLC-PHA 58.34 Lebensmitteltechnologie VZ AR 129 2020 6 730-740 11 |
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detailed small-scale characterization and scale-up of active yfp inclusion body production with escherichia coli induced by a tetrameric coiled coil domain |
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Detailed small-scale characterization and scale-up of active YFP inclusion body production with Escherichia coli induced by a tetrameric coiled coil domain |
| abstract |
During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37 °C to 15 °C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12 g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass. |
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During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37 °C to 15 °C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12 g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass. |
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During heterologous protein production with Escherichia coli, the formation of inclusion bodies (IBs) is often a major drawback as these aggregated proteins are usually inactive. However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37 °C to 15 °C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12 g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass. |
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However, different strategies for the generation of IBs consisting of catalytically active proteins have recently been described. In this study, the archaeal tetrameric coiled-coil domain of the cell-surface protein tetrabrachion was fused to a target reporter protein to produce fluorescent IBs (FIBs). As the cultivation conditions severely influence IB formation, the entire cultivation process resulting in the production of FIBs were thoroughly studied. First, the cultivation process was scaled down based on the maximum oxygen transfer capacity, combining online monitoring technologies for shake flasks and microtiter plates with offline sampling. The evaluation of culture conditions in complex terrific broth autoinduction medium showed strong oxygen limitation and leaky expression. Furthermore, strong acetate formation and pH changes from 6.5 to 8.8 led to sub-optimal cultivation conditions. However, in minimal Wilms-MOPS autoinduction medium, defined culture conditions and a tightly controlled expression were achieved. The production of FIBs is strongly influenced by the induction strength. Increasing induction strengths result in lower total amounts of functional protein. However, the amount of functional FIBs increases. Furthermore, to prevent the formation of conventional inactive IBs, a temperature shift from 37 °C to 15 °C is crucial to generate FIBs. Finally, the gained insights were transferred to a stirred tank reactor batch fermentation. Hereby, 12 g/L FIBs were produced, making up 43 % (w/w) of the total generated biomass.</subfield></datafield><datafield tag="650" ind1=" " ind2="7"><subfield code="a">Fluorescent protein</subfield><subfield code="2">Elsevier</subfield></datafield><datafield tag="650" ind1=" " ind2="7"><subfield code="a">Process characterization</subfield><subfield code="2">Elsevier</subfield></datafield><datafield tag="650" ind1=" " ind2="7"><subfield code="a">High-throughput technologies</subfield><subfield code="2">Elsevier</subfield></datafield><datafield tag="650" ind1=" " ind2="7"><subfield code="a">Online monitoring</subfield><subfield code="2">Elsevier</subfield></datafield><datafield tag="650" ind1=" " ind2="7"><subfield code="a">Active inclusion bodies</subfield><subfield code="2">Elsevier</subfield></datafield><datafield tag="650" ind1=" " ind2="7"><subfield code="a">Scale-up</subfield><subfield code="2">Elsevier</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Jäger, Vera D.</subfield><subfield code="4">oth</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Heyman, Benedikt</subfield><subfield code="4">oth</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Berg, Christoph</subfield><subfield code="4">oth</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Cürten, Christin</subfield><subfield code="4">oth</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Krauss, Ulrich</subfield><subfield code="4">oth</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Jaeger, Karl-Erich</subfield><subfield code="4">oth</subfield></datafield><datafield tag="700" ind1="1" ind2=" "><subfield code="a">Büchs, Jochen</subfield><subfield code="4">oth</subfield></datafield><datafield tag="773" ind1="0" ind2="8"><subfield code="i">Enthalten in</subfield><subfield code="n">Elsevier Science</subfield><subfield code="a">Saunders, Richard ELSEVIER</subfield><subfield code="t">Oral Squamous Cell Carcinoma in Three Related Kowari (Dasyuroides byrnei)</subfield><subfield code="d">2017</subfield><subfield code="g">Amsterdam [u.a.]</subfield><subfield code="w">(DE-627)ELV020602480</subfield></datafield><datafield tag="773" ind1="1" ind2="8"><subfield code="g">volume:129</subfield><subfield code="g">year:2020</subfield><subfield code="g">number:6</subfield><subfield code="g">pages:730-740</subfield><subfield code="g">extent:11</subfield></datafield><datafield tag="856" ind1="4" ind2="0"><subfield code="u">https://doi.org/10.1016/j.jbiosc.2020.02.003</subfield><subfield code="3">Volltext</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_USEFLAG_U</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">GBV_ELV</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">SYSFLAG_U</subfield></datafield><datafield tag="912" ind1=" " ind2=" "><subfield code="a">SSG-OLC-PHA</subfield></datafield><datafield tag="936" ind1="b" ind2="k"><subfield code="a">58.34</subfield><subfield code="j">Lebensmitteltechnologie</subfield><subfield code="q">VZ</subfield></datafield><datafield tag="951" ind1=" " ind2=" "><subfield code="a">AR</subfield></datafield><datafield tag="952" ind1=" " ind2=" "><subfield code="d">129</subfield><subfield code="j">2020</subfield><subfield code="e">6</subfield><subfield code="h">730-740</subfield><subfield code="g">11</subfield></datafield></record></collection>
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