Pedro Menvielle - Academia.edu (original) (raw)

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Papers by Pedro Menvielle

We have studied two different strategies in order to achieve a high-titer recombinant protein pro... more We have studied two different strategies in order to achieve a high-titer recombinant protein production in mammalian cell culture: improvement of recombinant clone by genetic engineering, and optimization of the cell culture media. In the biopharmaceutical industry, much attention has been given lately to the identification of a promoter from highly expressed endogenous genes and a suitable chromosomal location for the therapeutic gene integration. We analyzed protein expression profiles from Chinese Hamster Ovary (CHO) cells and cell culture supernatant in order to identify highly abundant proteins, secreted as well as intracellular proteins. On this way we were able to identify cyclophilin A protein, also known as Peptidyl-prolyl cis-trans isomerase A (PpiA), as a good candidate for a promoter activity study. Since CHO full genomic sequence was not yet available, we performed genome walking analysis, counting on the highly conservation of this gene amongst mammals, in order to sequence the PpiA gene and its 5' and 3' UTRs. We were not able to localize PpiA gene and obtain its promoter region sequence. Further studies are necessary in order to test the PpiA promoter activity in CHO cells, and evaluate this gene as a potential suitable chromosomal location for site-directed integration studies. The second approach involved the culture media optimization. We tested a sulfated polysaccharide Dextran sulfate 5.000 Da (DS) as a culture supplement. In this study, DS was used on anchorage-dependent CHO cells producing erythropoietin (EPO), as well as on HeLa cells, in order to investigate the effect of this molecule on anti-apoptotic and pro-survival cellular pathways. DS 5.000 Da treatment was shown to prolong the life of cells and increase productivity of EPO by 1.8-fold in comparison with controls. Furthermore, it was shown that DS inhibited apoptosis and caused a G0/G1 cell-cycle arrest, decreased p53 expression, and decreased amounts of phosphorylated ERK 1/2. DS treatment also resulted in an enhanced LC3-II/LC3-I ratio, and increased autophagosome formation seen by tagged-LC3. In addition, DS also increased expression of two hallmark Chaperone Mediated Autophagy (CMA) markers, HSC70 3 and LAMP2a suggesting possible CMA activation upon DS supplementation. Fluorescein-labeled DS 5.000 Da showed that DS was able to enter the cell, and colocalized with LC3 and HSC70. Our data showed that low doses of DS 5.000 Da may promote macroautophagy and CMA as pro-survival pathways, most likely in a cell typeindependent manner, suggesting that a better understanding and manipulation of phenomenon of autophagy could be of crucial importance in the bio-pharmaceutical industry, in particular in the field of recombinant protein production.

We have studied two different strategies in order to achieve a high-titer recombinant protein pro... more We have studied two different strategies in order to achieve a high-titer recombinant protein production in mammalian cell culture: improvement of recombinant clone by genetic engineering, and optimization of the cell culture media. In the biopharmaceutical industry, much attention has been given lately to the identification of a promoter from highly expressed endogenous genes and a suitable chromosomal location for the therapeutic gene integration. We analyzed protein expression profiles from Chinese Hamster Ovary (CHO) cells and cell culture supernatant in order to identify highly abundant proteins, secreted as well as intracellular proteins. On this way we were able to identify cyclophilin A protein, also known as Peptidyl-prolyl cis-trans isomerase A (PpiA), as a good candidate for a promoter activity study. Since CHO full genomic sequence was not yet available, we performed genome walking analysis, counting on the highly conservation of this gene amongst mammals, in order to sequence the PpiA gene and its 5' and 3' UTRs. We were not able to localize PpiA gene and obtain its promoter region sequence. Further studies are necessary in order to test the PpiA promoter activity in CHO cells, and evaluate this gene as a potential suitable chromosomal location for site-directed integration studies. The second approach involved the culture media optimization. We tested a sulfated polysaccharide Dextran sulfate 5.000 Da (DS) as a culture supplement. In this study, DS was used on anchorage-dependent CHO cells producing erythropoietin (EPO), as well as on HeLa cells, in order to investigate the effect of this molecule on anti-apoptotic and pro-survival cellular pathways. DS 5.000 Da treatment was shown to prolong the life of cells and increase productivity of EPO by 1.8-fold in comparison with controls. Furthermore, it was shown that DS inhibited apoptosis and caused a G0/G1 cell-cycle arrest, decreased p53 expression, and decreased amounts of phosphorylated ERK 1/2. DS treatment also resulted in an enhanced LC3-II/LC3-I ratio, and increased autophagosome formation seen by tagged-LC3. In addition, DS also increased expression of two hallmark Chaperone Mediated Autophagy (CMA) markers, HSC70 3 and LAMP2a suggesting possible CMA activation upon DS supplementation. Fluorescein-labeled DS 5.000 Da showed that DS was able to enter the cell, and colocalized with LC3 and HSC70. Our data showed that low doses of DS 5.000 Da may promote macroautophagy and CMA as pro-survival pathways, most likely in a cell typeindependent manner, suggesting that a better understanding and manipulation of phenomenon of autophagy could be of crucial importance in the bio-pharmaceutical industry, in particular in the field of recombinant protein production.