High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice

Abstract

When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES) has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i) there are no publicly available cloning vectors harboring a 2A peptide gene and (ii) comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required.

Figure 1. Construction of expression plasmids harboring DNA sequences encoding various 2A peptides flanked by multiple cloning sites.

(A) Schematic representation of cleavage occurring in a peptide translated in foot-and-mouth disease virus (FMDV). 1D, 2A and 2B indicate contiguous endogenous peptides translated in FMDV. The arrowhead indicates cleavage site. (B) DNA and corresponding amino acid sequences of various 2A peptides. Underlined sequences encode amino acids GSG, which were added to improve cleavage efficiency. P2A indicates porcine teschovirus-1 2A; T2A, Thoseaasigna virus 2A; E2A, equine rhinitis A virus (ERAV) 2A; F2A, FMDV 2A. (C) A schema showing a map for an expression plasmid harboring DNA sequences encoding a 2A peptide (upper) and a construct used in this study encoding 2A peptides flanked by NLS-EGFP and mCherry-CAAX. NLS and CAAX denote the nuclear localization sequence and membrane localization sequence, respectively. (D) Sequences of the multiple cloning sites and recognition sites of unique restriction endonucleases therein. Nucleotides in blue encode a P2A peptide.

Figure 2. P2A shows the highest cleavage efficiency in HEK293T cells.

HEK293T cells were transfected with the indicated plasmids. (A) WB analysis of cleavage efficiency of the 2As in HEK293T cells. The transfected cells were processed for WB 24 hr post-transfection. The cleavage efficiency was assessed using GFP and DsRed antibodies to decorate NLS-EGFP and mCherry-CAAX, respectively. Asterisks indicate two major byproducts. Anti-β actin antibody was used as a loading control. (B) Quantitation of cleavage efficiency of indicated 2As. Cleavage efficiency = cleaved form/(cleaved form+uncleaved form). The amount of each form was estimated from its band intensity on the Western blot measured by ImageJ software. The p value was determined by the two-tailed Student's t-test (n = 3). (C) Confocal microscopy of the transfected cells. Green signals in the nucleus that do not overlap with red signals indicate cleaved NLS-EGFP. Conversely, red signals in the plasma membrane that do not overlap with green signals represent cleaved mCherry-CAAX. In the overlay images, yellow signals derived from overlapping of green and red signals denote uncleaved NLS-EGFP-2A-mCherry-CAAX. The scale bar represents 20 µm.

Figure 3. P2A shows the highest cleavage efficiency in HT1080 cells.

(A–C) HT1080 cells were transfected with indicated plasmids and analyzed as described in Figure 2. The scale bar represents 20 µm.

Figure 4. P2A shows the highest cleavage efficiency in HeLa cells.

(A–C) HeLa cells were transfected with indicated plasmids and analyzed as described in Figure 2. The scale bar represents 20 µm.

Figure 5. P2A shows the highest cleavage efficiency in zebrafish embryos.

One- or two-cell stage embryos were injected with in vitro transcribed RNAs encoding the indicated proteins. (A) WB analysis revealing cleavage efficiency in the 2As in zebrafish embryos. The injected embryos at 24 hpf were processed for WB. Anti-β actin antibody was used as a loading control. (B) Quantitation of cleavage efficiency of the indicated 2As. The cleavage efficiency was calculated as depicted in Figure 2B. P value was determined by the two-tailed Student's t-test (n = 3). (C) Confocal microscopy of the retina in the injected embryos at 24 hpf. Non-overlapping green signals in the nucleus and red signals in the plasma membrane correspond to cleaved NLS-EGFP and mCherry-CAAS, respectively. Yellow signals in the overlay images signify uncleaved NLS-EGFP-2A-mCherry-CAAX. The scale bar represents 20 µm.

Figure 6. P2A shows the highest cleavage efficiency in the mouse liver.

Adult mice were injected with recombinant adenovirus encoding indicated proteins via the tail vein. (A) WB analysis revealing cleavage efficiency of the 2As in the mouse liver. The liver was extracted from the injected mice at 3 day post-injection (dpi) and processed for WB. Anti-β actin antibody was used as a loading control. (B) Quantitation of cleavage efficiency of the indicated 2As. The cleavage efficiency was calculated as depicted in Figure 2b. The p value was determined by the two-tailed Student's t-test (n = 3). (C) Confocal microscopy of liver section of the injected mice. The images were taken at 3 dpi. Green, red and yellow signals are as described in the legend for Figure 5C. The scale bar represents 20 µm.