A dual prokaryotic (E. coli) expression system (pdMAX)
Abstract In this study, we introduced an efficient subcloning and expression system with two inducible prokaryotic expression promoters, arabinose and lac, in a single plasmid in Escherichia coli. This pdMAX system is a manageable size at 5811 bp. The arabinose promoter unit allows for the expression of a FLAG-tagged protein, while the isopropyl-β-D-thiogalactoside (IPTG)-inducible unit allows for the expression of a Myc-tagged protein. An efficient subcloning (DNA insertion) system (iUnit) follows each promoter. The iUnit, based on a toxin that targets DNA topoisomerase of E. coli, allows for effective selection with arabinose or IPTG induction.Interestingly, the dual induction plasmid system shows limited protein expression. To analyze the expression of inserted genes, we performed an α-complementation assay, in which the α-peptide of the lacZ (β-galactosidase) gene is inserted in XL-10 E. coli cells. E. coli expressing the recombinant plasmid form blue colonies when plated on ampicillin/IPTG- or arabinose/X-gal-containing plates if the α-peptide was correctly inserted in-frame to produce the tagged protein. This assay system assesses whether the α-peptide was inserted at the restriction sites (EcoRV or SmaI), whether the inserted peptide was expressed, and whether the inserted α-peptide sequence was ligated in-frame to produce a tagged protein. Frameshifts result in no α-complementation and white colonies.With the dual promoter plasmid (pdMAX) system, expressed lacZ activity was significantly decreased comparing with the solo expression (pgMAX) system. Despite this disadvantage, we believe the pdMAX system is still useful for the analysis of distinct genes in E. coli, which will enable different types of expression analysis. Overall, the novel pdMAX system allows for efficient subcloning of two different genes. Furthermore, the pdMAX system could be used to induce and analyze the expression of two distinct genes and adopted to various types of prokaryotic gene expression analyses..
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Preprint| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
bioRxiv.org - (2021) vom: 15. Dez. Zur Gesamtaufnahme - year:2021 |
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| Sprache: |
Englisch |
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| Beteiligte Personen: |
Murakami, Manabu [VerfasserIn] |
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| Links: |
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| doi: |
10.1101/2020.07.23.217315 |
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| PPN (Katalog-ID): |
XBI018421075 |
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| 520 | |a Abstract In this study, we introduced an efficient subcloning and expression system with two inducible prokaryotic expression promoters, arabinose and lac, in a single plasmid in Escherichia coli. This pdMAX system is a manageable size at 5811 bp. The arabinose promoter unit allows for the expression of a FLAG-tagged protein, while the isopropyl-β-D-thiogalactoside (IPTG)-inducible unit allows for the expression of a Myc-tagged protein. An efficient subcloning (DNA insertion) system (iUnit) follows each promoter. The iUnit, based on a toxin that targets DNA topoisomerase of E. coli, allows for effective selection with arabinose or IPTG induction.Interestingly, the dual induction plasmid system shows limited protein expression. To analyze the expression of inserted genes, we performed an α-complementation assay, in which the α-peptide of the lacZ (β-galactosidase) gene is inserted in XL-10 E. coli cells. E. coli expressing the recombinant plasmid form blue colonies when plated on ampicillin/IPTG- or arabinose/X-gal-containing plates if the α-peptide was correctly inserted in-frame to produce the tagged protein. This assay system assesses whether the α-peptide was inserted at the restriction sites (EcoRV or SmaI), whether the inserted peptide was expressed, and whether the inserted α-peptide sequence was ligated in-frame to produce a tagged protein. Frameshifts result in no α-complementation and white colonies.With the dual promoter plasmid (pdMAX) system, expressed lacZ activity was significantly decreased comparing with the solo expression (pgMAX) system. Despite this disadvantage, we believe the pdMAX system is still useful for the analysis of distinct genes in E. coli, which will enable different types of expression analysis. Overall, the novel pdMAX system allows for efficient subcloning of two different genes. Furthermore, the pdMAX system could be used to induce and analyze the expression of two distinct genes and adopted to various types of prokaryotic gene expression analyses. | ||
| 700 | 1 | |a Murakami, Agnieszka M. |e verfasserin |4 aut | |
| 700 | 1 | |a Hirota, Kazuyoshi |e verfasserin |4 aut | |
| 700 | 1 | |a Itagaki, Shirou |e verfasserin |4 aut | |
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