A simple, dual direct expression plasmid system in prokaryotic and mammalian cells

Abstract We introduce a simple, dual direct cloning plasmid system (pgMAX-II) for gene expression analysis in both prokaryotic (Escherichia coli) and mammalian cells. This system, which uses a prokaryotic expression unit adapted from the pgMAX system and a mammalian promoter, is effective for subcloning using the DNA topoisomerase II toxin CcdB. Given that molecular biological cloning systems broadly rely on E. coli for rapid growth, the proposed concept may have wide applicability beyond mammalian cells.


Introduction
The standard process of creating mammalian transient expression plasmid constructs typically involves two steps: first, the desired gene is subcloned into a subcloning plasmid, such as pBluescript (Agilent Technologies, Santa Clara, CA, USA); then, the desired gene is converted from the subcloning plasmid to a mammalian expression plasmid, such as pcDNA3 (Thermo Fisher Scientific, Waltham, MA, USA; Conversion method) (1). In 2019, we developed a dual (Escherichia coli and mammalian) expression plasmid, pgMAX. This system enables efficient subcloning and expression in E. coli; moreover, following a simple deletion step of the prokaryotic promoter sequence with the rare-cutter restriction enzymes SwaI and PmeI and religation, mammalian expression can be achieved (Deletion method) (1).
In the present study, we established a dual expression plasmid (pgMAX-II) that does not require the deletion step of the original pgMAX system. Instead, pgMAX-II can be used as a prokaryotic expression vector with E. coli and can also be used directly as a mammalian expression vector (Direct method). Figure 1A presents an overview of the pgMAX plasmid system (1). The pgMAX plasmid has two functional components: the prokaryotic component for prokaryotic gene expression (lac promoter and lac operator) with an inhibitory unit (iUnit), containing CcdB for efficient subcloning (prokaryotic expression unit, Fig. 1); and the mammalian expression component comprising the cytomegalovirus (CMV) promoter and a poly-A tail (1,2).

Plasmid construction
A direct expression plasmid for both prokaryotic (E. coli) and mammalian expression requires a sequence that can be used as a prokaryotic promoter in E. coli and does not inhibit mammalian gene expression under the CMV promoter.
To develop the pgMAX-II system, the PCR-amplified enhanced green fluorescent protein (EGFP) gene was inserted into the EcoRV site of pgMAX. Then, the sequence between HindIII and the lac promoter (i.e. the isopropyl-β-D-thiogalactoside [IPTG]-inducible sequence) was mutated by PCR with degenerate oligo DNA (Fig. 1Bi). With this PCR-mutated prokaryotic promoter sequence, GFP fluorescence was observed (excitation wavelength: 470 nm, emission wavelength: 505 nm; Fig. 1Bii). Colonies exhibiting green fluorescence were selected and each plasmid clone was transiently transfected in human embryonic kidney (HEK)293T cells. One clone (out of 16) showed bright GFP fluorescence in both E. coli and HEK293T cells, which we named pgMAX-II/EGFP. Then, the EGFP gene was eliminated using EcoRV. The colonies were inoculated on Luria broth (LB) agar plates containing either ampicillin (amp) alone or amp and IPTG, to confirm the effect of IPTG and toxicity of CcdB protein (Fig. 1Biii). The recombinant group showed no growth on the LB plate containing IPTG, indicative of CcdB expression. This plasmid was named pgMAX-II (DNA alignment of the prokaryotic promoter region of pgMAX and pgMAX-II was shown in the supplementary information).

Effective subcloning and expression in E. coli and HEK cells
To confirm efficient cloning with pgMAX-II (i.e. successful recombination) and protein expression, the PCR-amplified α-peptide sequence of the lacZ (β-galactosidase) gene was inserted between the blunt-end sites of EcoRV of pgMAX-II (3,4). Following ligation and transformation, the recombinant clones were plated on LB agar containing amp, X-gal (0.004 mg/mL), and IPTG (for lac operon induction). After 16 h, the numbers of blue (α-complementation with sense-directed ligation of the DNA fragment) and white (antisense-directed ligation or no insertion of the DNA fragment) colonies were evaluated ( Fig. 2A). Nearly one-third (28.4%; 77 of 271 colonies) of colonies were blue, indicative of the high efficacy of the pgMAX-II plasmid for subcloning and protein expression in E. coli.
We further examined ligation of the PCR-amplified DsRed2 fragment (∼700 bp) and EGFP gene (∼700 bp). Replated clones containing DsRed2 or EGFP resulted in high expression of the respective fluorescent proteins (Fig. 2A). Figure 2B presents the results of PCR analysis of randomly selected clones after EGFP gene ligation in pgMAX-II. PCR analysis revealed successful subcloning (87.5%, 7 of 8 clones contained the desired insert).

Direct expression in mammalian cells using the pgMAX-II plasmid
To analyze mammalian expression using the pgMAX-II system, we evaluated the pgMAX-II plasmid containing the EGFP gene. After 48 h of plasmid DNA transfection in HEK293T cells, EGFP fluorescence was observed under a fluorescence microscope (excitation wavelength: 470 nm, emission wavelength: 505 nm). The pEGFP plasmid (positive control) containing the CMV promoter, EGFP gene, and poly-A sequence showed bright fluorescence (Fig. 2C). The pgMAX-II/EGFP plasmid containing the EGFP gene in the EcoRV site, which showed bright fluorescence in E. coli, was used directly (without DNA recombination or deletion of the prokaryotic unit) for transient transfection in HEK293T cells. EGFP fluorescence in HEK293T cells with pgMAX-II/EGFP was comparable with that of pEGFP-transfected cells. Using an ORCA imaging system (Hamamatsu, Shizuoka, Japan) to quantify EGFP fluorescence, the fluorescence intensity associated with transfection with pgMAX-II/ EGFP (1.39 ± 0.30 U, n = 9) was 52.9% of that with pEGFP (2.63 ± 0.36 U, n = 9; P < 0.05 vs. pEGFP; Fig. 2C). We also examined pgMAX-II plasmid containing DsRed2, and obtained similar results.
In conclusion, we established a simple, universal direct cloning plasmid system for gene expression in both prokaryotic (E. coli) and mammalian cells. This system (pgMAX-II) can be applied directly for expression analysis, for example, transient expression in mammalian cells. The highly efficient subcloning of this plasmid system is dependent on the toxicity of CcdB during DNA reproduction in E. coli (1, 2, 4). Interestingly, this toxic activity could be easily impacted by externally inserted DNA, resulting in a significant decrease in toxicity and colony formation. Because E. coli is used for complementary DNA cloning in various species, our system could have wide applicability for expression analyses.