A new dual prokaryotic (E. coli) and mammalian expression system (pgMAXs)
Abstract We introduce an efficient subcloning and expression plasmid system with two different modes (prokaryotic for expression in Escherichia coli with lac promoter and mammalian modes with cytomegalovirus promoter). The efficient subcloning (DNA insertion) is based upon a DNA topoisomerase II toxin-originated gene for effective selection with isopropyl-β-D-thiogalactoside (IPTG) induction. The new pgMAXs system is manageable size (4452 bp) and has also various types of protein tags (flag, myc, poly-histidine, Human influenza hemagglutinin, strep, and v5) for expression analysis. With pgMAXs system, various types of fluorescent proteins were subcloned and prtein expressions were confirmed. We also tried to identify epitope amino acid sequences for anti-calcium channel β2 antibody, by constructing epitope-library with DNaseI-partial digestion and subcloning into EcoRV site in pgMAXs. The new pgMAXs plasmid system enables highly efficient subcloning, simple expression in E. coli and that it has a simple deletion step of rare 8-nucleotide rare-cutter blunt-end enzymes for mammalian expression plasmid construction. Taken together, the pgMAXs system simplifies prokaryotic and mammalian gene expression analyses..
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Preprint| Erscheinungsjahr: |
2020 |
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| Erschienen: |
2020 |
| Enthalten in: |
bioRxiv.org - (2020) vom: 24. Mai Zur Gesamtaufnahme - year:2020 |
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| Sprache: |
Englisch |
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| Beteiligte Personen: |
Murakami, Manabu [VerfasserIn] |
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| Links: |
Volltext [kostenfrei] |
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| doi: |
10.1101/2020.05.21.108126 |
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| funding: |
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| PPN (Katalog-ID): |
XBI017929008 |
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| 245 | 1 | 0 | |a A new dual prokaryotic (E. coli) and mammalian expression system (pgMAXs) |
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| 520 | |a Abstract We introduce an efficient subcloning and expression plasmid system with two different modes (prokaryotic for expression in Escherichia coli with lac promoter and mammalian modes with cytomegalovirus promoter). The efficient subcloning (DNA insertion) is based upon a DNA topoisomerase II toxin-originated gene for effective selection with isopropyl-β-D-thiogalactoside (IPTG) induction. The new pgMAXs system is manageable size (4452 bp) and has also various types of protein tags (flag, myc, poly-histidine, Human influenza hemagglutinin, strep, and v5) for expression analysis. With pgMAXs system, various types of fluorescent proteins were subcloned and prtein expressions were confirmed. We also tried to identify epitope amino acid sequences for anti-calcium channel β2 antibody, by constructing epitope-library with DNaseI-partial digestion and subcloning into EcoRV site in pgMAXs. The new pgMAXs plasmid system enables highly efficient subcloning, simple expression in E. coli and that it has a simple deletion step of rare 8-nucleotide rare-cutter blunt-end enzymes for mammalian expression plasmid construction. Taken together, the pgMAXs system simplifies prokaryotic and mammalian 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 | |
| 773 | 0 | 8 | |i Enthalten in |t bioRxiv.org |g (2020) vom: 24. Mai |
| 773 | 1 | 8 | |g year:2020 |g day:24 |g month:05 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1101/2020.05.21.108126 |z kostenfrei |3 Volltext |
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