Triple gene expressions in yeast, Escherichia coli, and mammalian cells by transferring DNA fragments amplified from a mother yeast expression plasmid

Abstract

Escherichia coli, Saccharomyces cerevisiae, and mammalian culture cells are standard host organisms for genetic engineering and research, thus various plasmid vectors have been developed. However, the vectors are designed only for a single host owing to their host-specific genetic elements such as promoters and selection markers. In this study, we developed a yeast expression plasmid that enables the expression of the same gene in E.coli and mammalian cells via the transfer of PCR products amplified from the plasmid as a template. The yeast plasmid YHp26352 was constructed to contain the following regions sequentially: yeast TDH3 promoter (TDH3p), red fluorescent protein (eEmRFP), SV40 terminator (SVpA), E.coli origin (ori), ampicillin resistant gene (AmpR), mammalian cytomegalovirus promoter (CMVp), E.coli srlA promoter (srlAp), and yeast selection marker URA3, which expressed eEmRFP in yeast. To express eEmRFP in mammalian cells, an end-promoter DNA fragment encompassing the eEmRFP-SVpA-ori-AmpR-CMVp region was amplified by PCR and directly used for transfection to mammalian culture cells, resulting in gene expression in mammalian cells through non-homologous end joining. Homologous recombination-mediated circularization was carried out for E.coli cloning and expression by attaching a short overlapping sequence to the 5'-end of a PCR primer, which was used to amplify the eEmRFP-SVpA-ori-AmpR-CMVp-srlAp fragment, after which E.coli transformation was performed. Proof-of-concept experiments were performed by expressing GFP-fused human synaptobrevin VAMP1, and wild-type and codon-changed CLuc luciferase genes in yeast, E.coli, and HEK293cells. This is the first all-in-one plasmid applicable for expression in three host organisms.