Date Published: May 2, 2019
Publisher: Public Library of Science
Author(s): Manabu Murakami, Takayoshi Ohba, Agnieszka M. Murakami, Chong Han, Kenji Kuwasako, Shirou Itagaki, Alberto Amato.
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-β-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.
Numerous commercial expression plasmids exist, especially for mammalian transient expression. The process of mammalian transient expression of a desired gene has mainly relied on a two-step method: subcloning of the desired gene into a subcloning plasmid such as pBluescript (Agilent Technologies, Santa Clara, CA, USA), and subcloning the desired gene into a mammalian expression plasmid such as pcDNA3 (Thermo Fisher Scientific, Waltham, MA, USA) . Each cloning step is often troublesome, due to the low efficiency of DNA ligation. There are several methods for the first subcloning step, including the traditional blunt-end treatment of a polymerase chain reaction (PCR) product with T4 DNA polymerase, ligating a fragment into a blunt-end EcoRV site in a pBluescript plasmid, or restriction enzyme digestion and ligation. Developments in molecular biology have enabled other cloning methods, such as TA, TOPO and Gateway cloning, although these have relatively high costs and are dependent on PCR [2–5]. TA cloning uses Taq DNA polymerase to add a single adenosine-residue overhang to the 3′ end of the PCR product. As Taq DNA polymerase lacks 3′ to 5′ exonuclease proofreading activity, it has relatively low fidelity. Furthermore, a single adenosine overhang becomes degraded over time, which reduces the ligation efficiency. TOPO cloning uses topoisomerase to unwind and ligate the DNA. Gateway cloning has the benefit of a single recombination reaction that moves a section of DNA from one plasmid to another. For rapid construction of multiple DNA fragment ligations, both Gibson Assembly and Golden Gate Assembly are available, although they require specific enzymes [6, 7]. Another ligation method for multiple DNA fragments, the SLIC method, has also been established . This method is cost effective and dependent on PCR and T4 exonuclease activity.
In the present study, we established a new subcloning/expression plasmid (pgMAX-system). This plasmid enables simple and highly efficient subcloning of a desired gene with standard techniques in E. coli (insertion step and prokaryotic mode), and easy construction of a mammalian expression plasmid within a few days (deletion step: restriction with SwaI and PmeI and re-ligation; after the deletion step, the plasmid is in mammalian mode). The pgMAX plasmid contains several 8-nucleotide rare-cutter enzyme sites that are useful for achieving mammalian mode. In our analysis, the DsRed2-originating PCR fragment exhibited bright red fluorescence, indicating the establishment of a new, simple and efficient expression plasmid system.
We established a new subcloning and expression plasmid with two different modes (prokaryotic and mammalian modes). The new plasmid system enables highly efficient subcloning of a blunt-end DNA fragment, simple expression in E. coli and that it has a simple deletion step for mammalian expression plasmid construction.