Increased productivity of recombinant tissular plasminogen activator (t-PA) by butyrate and shift of temperature: a cell cycle phases analysis |
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Authors: | V Hendrick P Winnepenninckx C Abdelkafi O Vandeputte M Cherlet T Marique G Renemann A Loa G Kretzmer J Werenne |
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Institution: | (1) Laboratory of Animal Cell Biotechnology, Faculty of Sciences, Université Libre de Bruxelles, CP160/17, 50 Av. F.D. Roosevelt, 1050 Brussels, Belgium;(2) Laboratory of Animal Cell Biotechnology, Faculty of Sciences, Université Libre de Bruxelles, CP160/17, 50 Av. F.D. Roosevelt, 1050 Brussels, Belgium;(3) Institut für Technische Chemie, University of Hannover, Germany |
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Abstract: | Directed control of cell metabolism by a modification of the physicochemical conditions (presence of Na-butyrate and modification
of the temperature) was used to modulate the productivity of human recombinant tissular plasminogen activator (t-PA) expressed
under control of SV40 promoter in Chinese Hamster Ovary (CHO) cell lines. We showed that both by adding Na-butyrate or lowering
temperature from 37 °C to 32 °C there is an increase in the amount of t-PA excreted, while cell growth is significantly reduced.
The treatments also increased the intracellular amount of t-PA. We measured the distribution of cell cycle phases by cytometry
and used a modification of the equations of Kromenaker and Srienc (1991, 1994 a, b) to analyse the intracellular t-PA production
rate in the different cell cycle phases. Intracellular t-PA was shown to accumulate in G1 phase in all conditions (at 37 °C,
at 32 °C and in presence of butyrate). Moreover, we have shown that the distribution of the time cells treated by butyrate
are maintained in the G1cell cycle phase is significantly increased. t-PA produced in the different cell culture conditions
tested was analysed by zymogram and western blotting: neither butyrate, neither the shift of temperature changed significantly
the overall quality of the protein. The N-glycan patterns of recombinant human t-PA was also analysed with carbohydrate-specific
lectins. Butyrate caused a transitory increase in N-linked complex high-mannose oligosaccharides without any effect on the
sialic acid content of t-PA.
This revised version was published online in July 2006 with corrections to the Cover Date. |
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Keywords: | cell cycle CHO cells Kromenaker and Srienc equations Na-butyrate Temperature shift t-PA production t-PA glycosylation |
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