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A simple and dual expression plasmid system in prokaryotic (E. coli) and mammalian cells.

ABSTRACT: We introduce a simple and universal cloning plasmid system for gene expression in prokaryotic (Escherichia coli) and mammalian cells. This novel system has two expression modes: the (subcloning) prokaryotic and mammalian modes. This system streamlines the process of producing mammalian gene expression plasmids with desired genes. The plasmid (prokaryotic mode) has an efficient selection system for DNA insertion, multiple component genes with rare restriction sites at both ends (termed "units"), and a simple transformation to mammalian expression mode utilizing rare restriction enzymes and re-ligation (deletion step). The new plasmid contains the lac promoter and operator followed by a blunt-end EcoRV recognition site, and a DNA topoisomerase II toxin-originated gene for effective selection with isopropyl-?-D-thiogalactoside (IPTG) induction. This system is highly efficient for the subcloning of blunt-end fragments, including PCR products. After the insertion of the desired gene, protein encoded by the desired gene can be detected in E. coli with IPTG induction. Then, the lac promoter and operator are readily deleted with 8-nucleotide rare-cutter blunt-end enzymes (deletion step). Following re-ligation and transformation, the plasmid is ready for mammalian expression analysis (mammalian mode). This idea (conversion from prokaryotic to mammalian mode) can be widely adapted. The pgMAX system overwhelmingly simplifies prokaryotic and mammalian gene expression analyses.

SUBMITTER: Murakami M

PROVIDER: S-EPMC6497378 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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A simple and dual expression plasmid system in prokaryotic (E. coli) and mammalian cells.

Murakami Manabu M Ohba Takayoshi T Murakami Agnieszka M AM Han Chong C Kuwasako Kenji K Itagaki Shirou S

PloS one 20190502 5

We introduce a simple and universal cloning plasmid system for gene expression in prokaryotic (Escherichia coli) and mammalian cells. This novel system has two expression modes: the (subcloning) prokaryotic and mammalian modes. This system streamlines the process of producing mammalian gene expression plasmids with desired genes. The plasmid (prokaryotic mode) has an efficient selection system for DNA insertion, multiple component genes with rare restriction sites at both ends (termed "units"), ...[more]

PMID: 31048860

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Project description:BackgroundInsulin-like growth factor binding protein-3 receptor (IGFBP-3R) (Transmembrane protein 219 [TMEM219]) binds explicitly to IGFBP-3 and exerts its apoptotic and autophagy signalling pathway. Constructing a Henrietta Lacks (HeLa) h6-TMEM219 cell characterize the therapeutic potent of TMEM219 that could interrupt the IGFBP-3/TMEM219 pathway, in cancer treatment and destructive cell illnesses such as diabetes and Alzheimer's.Materials and methodsFirst, to develop stable overexpressed HeLa h6-TMEM219 cells, and Escherichia coli BL21 (DE3) with high IGFBP-3R expression, the purchased pcDNA3.1-h6-TMEM219 plasmid was transformed and integrated using CaCl2 and chemical transfection reagents, respectively. The pcDNA3.1-h6-TMEM219 transfection and protein expression was evaluated by the polymerase chain reaction (PCR), western blotting, and flow cytometry. Following the induction of h6-TMEM219 expression, a protein was purified using Ni-NTA chromatography and evaluated by the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).ResultsThe 606 base pairs sequence in PCR outcomes confirmed successful pcDNA3.1-h6-TMEM219 transformation in E. Coli BL21 and integration into the HeLa genome. The analysis of protein samples from induced E. Coli BL21 and purified protein demonstrate a band of approximately 22 kDa on SDS-PAGE. Moreover, besides western blot analysis, flow cytometry findings illustrate approximately 84% of transfected HeLa cells (HeLa h6-TMEM219) overexpressed h6-TMEM219 on their surface.ConclusionWe designed a new experiment in the h6-TMEM219 expression procedure in both eukaryotic and prokaryotic hosts. All of our results confirm appropriate transformation and transfection and importantly, approve h6-TMEM 219 membrane expression. Finally, the HeLa h6-TMEM219 cells and the newly purified h6-TMEM219 leverage new studies for molecular diagnostic studies and characterize the therapeutic agents against IGFBP-3/TMEM219 signalling pathway in devastating illnesses in vitro and in vivo.

Dataset Information

A simple and dual expression plasmid system in prokaryotic (E. coli) and mammalian cells.

Publications

A simple and dual expression plasmid system in prokaryotic (E. coli) and mammalian cells.

Similar Datasets

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