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. 2001 Jul;36(1-3):71–83. doi: 10.1023/A:1014088919546

Increased productivity of recombinant tissular plasminogen activator (t-PA) by butyrate and shift of temperature: a cell cycle phases analysis

V Hendrick 1,✉, P Winnepenninckx 2, C Abdelkafi 2, O Vandeputte 2, M Cherlet 2, T Marique 2, G Renemann 3, A Loa 3, G Kretzmer 3, J Werenne 2
PMCID: PMC3449664  PMID: 19003317

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.

Keywords: cell cycle, CHO cells, Kromenaker and Srienc, equations, Na-butyrate, Temperature shift, t-PA production, t-PA glycosylation

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