Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
PLoS One. 2010 Dec 28;5(12):e14408. doi: 10.1371/journal.pone.0014408.
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. 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 FINDINGS: In 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/SIGNIFICANCE: Our 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.
尽管 293T 细胞被广泛用于表达转染质粒载体中的蛋白质，但高水平表达的分子基础仍有待了解。我们最近发现前列腺癌细胞系 PC3 在蛋白质表达方面与 293T 一样高效。本研究旨在破译这两种细胞系中高水平表达的分子基础。
方法/主要发现：在对不同细胞系进行血小板衍生生长因子-B（PDGF-B）、β-半乳糖苷酶（β-gal）和绿色荧光蛋白（GFP）从质粒载体高效表达的调查中，发现 PC3 的表达水平比骨转移前列腺癌细胞系 PC3BM 和许多其他细胞系高 5-50 倍。此外，报告基因在 PC3 中的转染效率和表达水平与 293T 相当。比较分析表明，这两种细胞系中报告基因的高水平表达是由于翻译效率的提高。虽然通过正丁醇显著降低报告基因表达来揭示的磷脂酸（PA）介导的 mTOR 激活主要导致 PC3 中的高表达，但涉及 PA、PI3K/Akt 和 ERK1/2 的多种途径似乎都有助于 293T 中大量报告基因的表达。因此，通过多种途径协同激活 mTOR 在 293T 中实现的翻译上调程度可以通过其在 PC3 中主要通过 PA 途径的激活来实现。
结论/意义：我们的研究表明，两种细胞系中通过 mTOR 激活的翻译上调来实现蛋白质的高水平表达，通过不同的信号通路，而 PC3 在重组蛋白表达方面与 293T 一样高效。此外，PC3 具有优势，因为可以通过向培养基中简单添加正丁醇来调节蛋白质的表达水平。
