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

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.


Introduction
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) [1]. 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 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 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][3][4][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 [8]. This method is cost effective and dependent on PCR and T4 exonuclease activity.
Nevertheless, it is usually necessary to first transfer the desired gene into a subcloning plasmid. After successful subcloning, the desired gene is treated by restriction enzyme digestion, agarose gel electrophoresis, DNA purification from an agarose gel fragment, and ligation into an expression vector. Otherwise, the subcloned genes can be used for further PCR-based plasmid construction. Here, we report a new type of plasmid, pgMAX, which enables highly efficient subcloning, expression of the desired gene with IPTG induction in Escherichia coli, and the simple and rapid construction of mammalian expression plasmids.

Cell culture and transfection
Cell culture and lipofection were performed as described previously [11]. Human embryonic kidney cells (HEK293, ATCC CRL 1573) were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum. Exponentially growing cells were plated onto 35-mm dishes, and lipofection was performed using commercially prepared lipofectamine (Invitrogen).

RNA isolation and reverse transcription (RT-PCR) analysis
Total RNA was isolated from the HEK 293T cells using an RNeasy extraction kit (Qiagen Inc., Valencia, CA, USA). The reverse transcription reaction was performed in a solution of 10 pmol oligo-dT primer, 1 μg RNA, 1 x first strand cDNA buffer (Life Technologies, Rockville, MD, USA), 10 mM dithiothreitol, 0.4 mM dNTPs and 200 U Superscript III (Thermo Fisher Scientific), in a volume of 20 μl, at 42˚C for 45 min. DsRed2 -specific sequences were amplified by PCR (30 cycles) with DsRed2for andDsRed2rev primers, which generated a PCR product of 699 bp for the comparative RT-PCR analysis.

Western blot analysis
Cell lysates from cell lines were prepared using TNE buffer in the presence of protease inhibitor cocktails (Roche Pharma, Basel, Switzerland). Lysates (100 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 15%) and blotted with the following antibodies: anti-Flag (Sigma-Aldrich, St. Louis, MO, USA), anti-DsRed (Arigo Biolaboratories Corp., Hsinchu City, Taiwan) and anti-GAPDH (Abcam Inc., Cambridge, UK) and were then incubated with alkaline phosphatase-conjugated secondary antibodies. The results were visualized by a colorimetric reaction using Western Blue Stabilized Substrate for Alkaline Phosphatase (Promega, Madison, WI, USA).
For Western blot analysis using E. coli, 0.5 ml of the culture medium (LB medium at 37˚C, 12-16 h) was harvested and resuspended in 100 μl lysis buffer (1 mM EDTA, 1 mg/ml lysozyme) and incubated at room temperature for 15 min. Aliquots (10 μl) of the homogenate from each clone were resolved by 15% SDS-PAGE and subjected to Western blotting. A commercially available polyclonal antibody specific for the Ca 2+ channel β 2 subunit (Sigma-Aldrich) was used, followed by a secondary anti-rabbit IgG antibody conjugated to alkalinephosphatase (Promega, Madison, WI, USA).

Statistics
Data are expressed as the means ± the standard error of the mean. Prior to statistical analyses, data were analyzed with the Shapiro-Wilk test. After confirmation of a normal distribution, statistical differences were further determined by Student's t-test or an analysis of variance with a Bonferroni post hoc test. P < 0.05 was considered to indicate statistical significance.  The pgMAX promotor has two functional components, prokaryotic and mammalian expression. The promoter is composed of a CMV promoter and IRES-puromycin N-acetyl-transferase gene (green arrow) with a poly A tail (pink arrow) (mammalian units I and II). The restriction enzyme (SwaI, PmeI, EcoRI, EcoRV, XhoI and NotI) sites are indicated. Oligo DNA for PCR screening is also indicated (pgMAXfor).

Plasmid construction
https://doi.org/10.1371/journal.pone.0216169.g001 A lac promoter and lac operator containing SwaI and PmeI restriction sites are under the control of the CMV promoter. At the PmeI site, a Kozak sequence followed by a Flag protein-coding sequence was inserted. A blunt-end DNA fragment can be inserted into the EcoRV bluntend site within the multiple cloning site, which also contains an inhibitory unit (iUnit).

Simple subcloning and expression in prokaryotic mode
First, we examined ligation of the PCR-amplified DsRed2 fragment (ca. 700 bp) into the EcoRV site of pgMAX. After 16 h of incubation on LB agar plates containing ampicillin (150 μg/ml) and IPTG (1 mM for lac operon induction), colonies were observed under green light (excitation wavelength 563 nm) through a filter set (emission wavelength 582 nm). Some colonies showed bright DsRed2-origin fluorescence (Fig 2A, red arrows). Colonies with red fluorescence were inoculated onto LB plates supplemented with ampicillin and IPTG for 16 h. Inoculated colonies containing the DsRed2 insert in the sense direction following the lac promoter exhibited bright red fluorescence (Fig 2B, double red arrows, pgMAX/DsRed).
As a next step, ligation of the PCR-amplified EGFP fragment (ca. 700 bp) was examined ( Fig 2C, green arrows). EGFP inserted pgMAX exhibited green fluorescence under blue light (excitation wavelength 470 nm) through a filter set (emission wavelength 505 nm). Because of the background fluorescence of E. coli, EGFP containing colonies were not as apparent as the DsRed2 containing colonies. Inoculated EGFP colonies exhibited bright green fluorescence (Fig 2D, double green arrows, pgMAX/EGFP).
As DsRed2 and EGFP are relatively short genes, we next examined Bgeo, which is a combination of β-galactosidase and the neomycin phosphotransferase gene (3.0 kb length). A bluntend Bgeo fragment was inserted into the EcoRV site of pgMAX. The constructed pgMAX/ Bgeo showed obvious blue colonies on a plate containing ampicillin, IPTG and Xgal (0.004 mg/ml), indicating the expression of β-galactosidase (Fig 2E blue arrows).
Among all of the 44 randomly picked colonies on the LB agar plates containing ampicillin and IPTG, 42 colonies contained the insert (DsRed2: 8 of 8, EGFP: 12 of 12, Bgeo: 20 of 22 colonies) with an approximately 90% success rate (S2 Table). Using fluorescence (DsRed2 or EGFP) or blue-white (X-gal) selection, all colonies (DsRed2: 8 of 8, EGFP: 8 of 8, Bgeo: 8 of 8 colonies) contained the expected inserts with the desired sense-direction of the lac promoter. Taken together, our data demonstrate that pgMAX is a simple and universal cloning plasmid system for subcloning and prokaryotic (E. coli) gene expression.

Simple preparation and expression for mammalian mode
To analyze mammalian expression using the pgMAX system, the pgMAX plasmid with the DsRed2 gene was evaluated. Colonies with red fluorescence were selected and grown in LB medium supplemented with ampicillin (150 μg/ml) for 16 h, and plasmid DNA was purified using standard techniques. Insertion of the DsRed2 fragment was confirmed with restriction enzymes (EcoRI and XhoI) and PCR with specific primers (pgMAXfor and DsRed2rev). The purified plasmid DNAs of these red colonies were further restricted with SwaI and PmeI and re-ligated to delete the lac promoter unit (SwaI-lac promoter-lac operator-PmeI sequence), before being transformed with standard competent cells and cultured for 16 h at 37˚C. The presence of the plasmid insert and the deletion of the lac promoter unit (combined sequence of the lac promoter and operator) were confirmed by PCR with specific primers (S1A  As another control, the commercially available pTarget plasmid (Promega) was also used for expression analysis. The PCR-amplified DsRed2 fragment was inserted into the EcoRV site of pTarget with blue-white selection. pTarget with the DsRed2 gene exhibited limited visible red DsRed2-related fluorescence was further measured with the Orca system, confirming comparable fluorescence intensity with DsRed2 plasmid transfection from day 1 to day 3 (87.4 ± 0.8 and 82.2 ± 1.4 U, DsRed2 and pgMAX/DsRed, respectively, n = 8, p < 0.05 vs. negative control, Fig 2B), while limited fluorescence was observed with pTarget containing DsRed2 (15.9 ± 3.5 U, n = 8, p < 0.05 vs. DsRed2 or pgMAX/Red). The overall fluorescence intensity data are available in S3 Table). Additionally, expression of the DsRed2 gene was evaluated by comparative RT-PCR (Fig 3C). DsRed2 (lane D) and pgMAX/Red (lane M) which showed comparable expression of the DsRed2 gene in HEK293T cells. To examine the universality of pgMAX/Red expression, transient expression of pgMAX/Red was examined in PC3, DU145 and KK47 cells. PC3 and DU145 are prostate cancer cell lines, while KK47 is a bladder cancer cell line. In all three of these cancer cell lines, pgMAX/Red exhibited fairly detectable red fluorescence (S1 Fig). Furthermore, the expression of Flag-tagged DsRed2 was confirmed with anti-Flag and anti-DsRed antibodies (Fig 3D) (the densitometric analysis results are shown in S4 Table). Additionally, puromycin (2 μM) selection was applied after transfection (S2 Fig). In negative control HEK293T cells (without pgMAX plasmid transfection), 24-h puromycin selection resulted in the death of over 90% of the cells, whereas most of the DsRe-d2-expressing cells survived (S2 Fig). Taken together, the pgMAX plasmid resulted in highly efficient subcloning with prokaryotic expression, easy construction of mammalian expression plasmid vector, and highly efficient selection using the IRES-puromycin resistance gene in cells with the desired gene expression [12].
Additionally, we examined prokaryotic expression of the rabbit voltage-dependent calcium channel β2a subunit using the pgMAX plasmid (S3 Fig). The rabbit β2a subunit cDNA was divided into four fragments (domains) and subcloned into the EcoRV site of pgMAX (S5 Table). The full-length cDNA of the β2a subunit was also subcloned into the same site. The construct was readily transformed into E. coli and selected using ampicillin and IPTG. Western blot analysis was performed using a commercially available polyclonal antibody specific for the Ca 2+ channel β2 subunit, which recognized domain D, indicating that the antibody recognized the C-terminal domain of the gene, and that the pgMAX system can be widely adapted (data for the densitometric analysis are shown in S6 Table).

Discussion
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.
The pgMAX plasmid may have several disadvantages. It has a relatively large size (6125 bp). Empirically, the insertion of a DNA fragment into a large plasmid is difficult. Therefore, it may be possible to experience difficulty with the first subcloning step (prokaryotic mode), especially when the insert is large. Furthermore, it is often difficult to produce a sufficient amount of DNA with a large plasmid. It is also important to note that pgMAX requires inframe ligation into the EcoRV site. In addition, if the desired gene displays toxicity in E. coli, it might be possible to obtain no colonies with the desired insert. In spite of these disadvantages, we must emphasize the overwhelming advantages of the pgMAX system, including highly efficient subcloning, protein expression in E. coli, simple construction of mammalian expression plasmids, and efficient and rapid (within 24 h) selection with puromycin. As expressed proteins in E. coli could be used for immunoblot analysis [13], this plasmid system may be useful for library construction for expression cloning. Puromycin could also be used to establish stable expression cell lines. We also examined direct subcloning of PCR products (without gel purification), such as the TA cloning system, with no success (S2 Table).
To our knowledge, the pTargetT mammalian expression vector (Promega) uses a similar concept to a prokaryotic promoter for efficient blue-white selection and a CMV-related promoter for mammalian expression, although the prokaryotic promoter is inserted in the antisense direction. The pTargetT vector functions as both a subcloning and a mammalian expression plasmid. It does not require a deletion step. In our hands, the pTargetT system resulted in simple ligation and prokaryotic expression was not detectable because of the anti-sense direction of the lac promoter, and the level of eukaryotic expression was limited (Fig 3A, pTarget/ Red).
On the other hand, pgMAX has a high ligation success rate (over 90% in our experience, S2 Table). It should be noted that expression of the desired gene product was detectable at the prokaryotic level (Fig 2A, 2B and 2C). It is also important to note that the pgMAX system uses only traditional molecular biology apparatuses and reagents. It does not require special substances, which TOPO cloning does [4]. In addition, the pgMAX system could be made to be even shorter (4454 bp) if the IRES-puromycin resistant gene (1661 bp) is deleted.
In the present study, we used an iUnit originating from CcdB, a toxin targeting the essential DNA gyrase of E. coli [14]. As iUnit selection is quite effective for fragment ligation, pgMAX has significant superiority over classical DNA expression systems with its prokaryotic and mammalian expression modes, as the expressed protein can be examined after the fragment ligation. For simple plasmid construction, we inserted rare restriction enzyme sites (mainly 8-nucleotide rare-cutter enzymes) at both ends of a functional gene, which we call a "unit." This idea could be widely useful for other types of plasmid construction.
Since the discovery of restriction enzymes and DNA ligase, a number of plasmids have been established [15,16]. Considering the use of restriction enzymes, the principal idea for the pgMAX plasmid is not novel. Despite this similarity, as we mainly inserted 8-nucleotide rarecutter enzyme sites (which should be cut every 4 8 bp) at each end of a gene (lac promoter and operator, iUnit, IRES-puromycin resistance gene), each unit can be easily handled. Taken together, our results indicate that the pgMAX plasmid system enables the simple and easy expression analysis of genes due to its rare-cutter sites.

Conclusion
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.
Supporting information S1