Abstract
BackgroundThough 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines.Methodology/Principal FindingsIn a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B), β-galactosidase (β-gal) and green fluorescent protein (GFP) from plasmid vectors, PC3 was found to express at 5–50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA)-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational up-regulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3.Conclusions/SignificanceOur studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to the culture medium.
Highlights
Gene expression in mammalian cells can be regulated at a single or multiple levels involving chromatin structure, transcription, post-transcription and translation leading to different genes being expressed at widely varying levels in a cell type-specific manner or in the same cell
Conclusions/Significance: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mammalian target of rapamycin (mTOR) activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression
Both RNA (2–3 fold) and protein (20–50 fold) levels of platelet-derived growth factorB (PDGF-B) were higher in PC3 and 293T when compared to those in HeLa and PC3BM suggesting that the translational efficiency of PDGF-B mRNA in these cell lines is .10-fold than in other cells
Summary
Gene expression in mammalian cells can be regulated at a single or multiple levels involving chromatin structure, transcription, post-transcription and translation leading to different genes being expressed at widely varying levels in a cell type-specific manner or in the same cell. The human embryonic kidney-derived HEK293 cells [1] exhibit very high transfection efficiency and express the recombinant proteins at high levels [2,3]. These cells were further modified by stable expression of the SV40 large T antigen generating the HEK293T (293T) cell line [4] which allows high level expression of proteins through episomal amplification of plasmids that contain SV40 origin of replication. Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines
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