Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system
Background Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The c...
Ausführliche Beschreibung
Autor*in: |
Shariati, Fatemeh Sadat [verfasserIn] |
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E-Artikel |
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Englisch |
Erschienen: |
2021 |
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Anmerkung: |
© The Author(s) 2021 |
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Übergeordnetes Werk: |
Enthalten in: Microbial cell factories - London : Biomed Central, 2002, 20(2021), 1 vom: 11. Dez. |
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Übergeordnetes Werk: |
volume:20 ; year:2021 ; number:1 ; day:11 ; month:12 |
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DOI / URN: |
10.1186/s12934-021-01711-2 |
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Katalog-ID: |
SPR050348868 |
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520 | |a Background Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27). Results Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze–thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking. Conclusion The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. This system can be investigated for other recombinant proteins expressed in E. coli with a potential capability for automation and use at larger scales. | ||
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700 | 1 | |a Norouzian, Dariush |4 aut | |
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10.1186/s12934-021-01711-2 doi (DE-627)SPR050348868 (SPR)s12934-021-01711-2-e DE-627 ger DE-627 rakwb eng Shariati, Fatemeh Sadat verfasserin aut Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2021 Background Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27). Results Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze–thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking. Conclusion The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. This system can be investigated for other recombinant proteins expressed in E. coli with a potential capability for automation and use at larger scales. Dicistronic expression system (dpeaa)DE-He213 Rapid screening (dpeaa)DE-He213 Self-inducible expression system (dpeaa)DE-He213 Enhanced green fluorescent protein (dpeaa)DE-He213 Norouzian, Dariush aut Valizadeh, Vahideh aut Ahangari Cohan, Reza (orcid)0000-0002-0490-4184 aut Keramati, Malihe aut Enthalten in Microbial cell factories London : Biomed Central, 2002 20(2021), 1 vom: 11. Dez. (DE-627)355987651 (DE-600)2091377-1 1475-2859 nnns volume:20 year:2021 number:1 day:11 month:12 https://dx.doi.org/10.1186/s12934-021-01711-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 20 2021 1 11 12 |
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10.1186/s12934-021-01711-2 doi (DE-627)SPR050348868 (SPR)s12934-021-01711-2-e DE-627 ger DE-627 rakwb eng Shariati, Fatemeh Sadat verfasserin aut Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2021 Background Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27). Results Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze–thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking. Conclusion The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. This system can be investigated for other recombinant proteins expressed in E. coli with a potential capability for automation and use at larger scales. Dicistronic expression system (dpeaa)DE-He213 Rapid screening (dpeaa)DE-He213 Self-inducible expression system (dpeaa)DE-He213 Enhanced green fluorescent protein (dpeaa)DE-He213 Norouzian, Dariush aut Valizadeh, Vahideh aut Ahangari Cohan, Reza (orcid)0000-0002-0490-4184 aut Keramati, Malihe aut Enthalten in Microbial cell factories London : Biomed Central, 2002 20(2021), 1 vom: 11. Dez. (DE-627)355987651 (DE-600)2091377-1 1475-2859 nnns volume:20 year:2021 number:1 day:11 month:12 https://dx.doi.org/10.1186/s12934-021-01711-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 20 2021 1 11 12 |
allfields_unstemmed |
10.1186/s12934-021-01711-2 doi (DE-627)SPR050348868 (SPR)s12934-021-01711-2-e DE-627 ger DE-627 rakwb eng Shariati, Fatemeh Sadat verfasserin aut Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2021 Background Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27). Results Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze–thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking. Conclusion The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. This system can be investigated for other recombinant proteins expressed in E. coli with a potential capability for automation and use at larger scales. Dicistronic expression system (dpeaa)DE-He213 Rapid screening (dpeaa)DE-He213 Self-inducible expression system (dpeaa)DE-He213 Enhanced green fluorescent protein (dpeaa)DE-He213 Norouzian, Dariush aut Valizadeh, Vahideh aut Ahangari Cohan, Reza (orcid)0000-0002-0490-4184 aut Keramati, Malihe aut Enthalten in Microbial cell factories London : Biomed Central, 2002 20(2021), 1 vom: 11. Dez. (DE-627)355987651 (DE-600)2091377-1 1475-2859 nnns volume:20 year:2021 number:1 day:11 month:12 https://dx.doi.org/10.1186/s12934-021-01711-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 20 2021 1 11 12 |
allfieldsGer |
10.1186/s12934-021-01711-2 doi (DE-627)SPR050348868 (SPR)s12934-021-01711-2-e DE-627 ger DE-627 rakwb eng Shariati, Fatemeh Sadat verfasserin aut Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2021 Background Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27). Results Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze–thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking. Conclusion The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. This system can be investigated for other recombinant proteins expressed in E. coli with a potential capability for automation and use at larger scales. Dicistronic expression system (dpeaa)DE-He213 Rapid screening (dpeaa)DE-He213 Self-inducible expression system (dpeaa)DE-He213 Enhanced green fluorescent protein (dpeaa)DE-He213 Norouzian, Dariush aut Valizadeh, Vahideh aut Ahangari Cohan, Reza (orcid)0000-0002-0490-4184 aut Keramati, Malihe aut Enthalten in Microbial cell factories London : Biomed Central, 2002 20(2021), 1 vom: 11. Dez. (DE-627)355987651 (DE-600)2091377-1 1475-2859 nnns volume:20 year:2021 number:1 day:11 month:12 https://dx.doi.org/10.1186/s12934-021-01711-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 20 2021 1 11 12 |
allfieldsSound |
10.1186/s12934-021-01711-2 doi (DE-627)SPR050348868 (SPR)s12934-021-01711-2-e DE-627 ger DE-627 rakwb eng Shariati, Fatemeh Sadat verfasserin aut Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system 2021 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s) 2021 Background Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27). Results Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze–thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking. Conclusion The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. This system can be investigated for other recombinant proteins expressed in E. coli with a potential capability for automation and use at larger scales. Dicistronic expression system (dpeaa)DE-He213 Rapid screening (dpeaa)DE-He213 Self-inducible expression system (dpeaa)DE-He213 Enhanced green fluorescent protein (dpeaa)DE-He213 Norouzian, Dariush aut Valizadeh, Vahideh aut Ahangari Cohan, Reza (orcid)0000-0002-0490-4184 aut Keramati, Malihe aut Enthalten in Microbial cell factories London : Biomed Central, 2002 20(2021), 1 vom: 11. Dez. (DE-627)355987651 (DE-600)2091377-1 1475-2859 nnns volume:20 year:2021 number:1 day:11 month:12 https://dx.doi.org/10.1186/s12934-021-01711-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 20 2021 1 11 12 |
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Shariati, Fatemeh Sadat |
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Shariati, Fatemeh Sadat misc Dicistronic expression system misc Rapid screening misc Self-inducible expression system misc Enhanced green fluorescent protein Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system |
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Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system Dicistronic expression system (dpeaa)DE-He213 Rapid screening (dpeaa)DE-He213 Self-inducible expression system (dpeaa)DE-He213 Enhanced green fluorescent protein (dpeaa)DE-He213 |
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rapid screening of high expressing escherichia coli colonies using a novel dicistronic-autoinducible system |
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Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system |
abstract |
Background Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27). Results Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze–thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking. Conclusion The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. This system can be investigated for other recombinant proteins expressed in E. coli with a potential capability for automation and use at larger scales. © The Author(s) 2021 |
abstractGer |
Background Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27). Results Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze–thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking. Conclusion The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. This system can be investigated for other recombinant proteins expressed in E. coli with a potential capability for automation and use at larger scales. © The Author(s) 2021 |
abstract_unstemmed |
Background Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27). Results Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze–thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking. Conclusion The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. This system can be investigated for other recombinant proteins expressed in E. coli with a potential capability for automation and use at larger scales. © The Author(s) 2021 |
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Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system |
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The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27). Results Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze–thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking. Conclusion The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. 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