Increased productivity of recombinant tissular plasminogen activator (t-PA) by butyrate and shift of temperature: a cell cycle phases analysis.
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 degrees C to 32 degrees 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 degrees C, at 32 degrees 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.
Hendrick
V
V
Laboratory of Animal Cell Biotechnology, Faculty of Sciences, Université Libre de Bruxelles, CP160/17, 50 Av. F.D. Roosevelt, 1050, Brussels, Belgium, vincianne.hendrick@compaqnet.be.
Winnepenninckx
P
P
Abdelkafi
C
C
Vandeputte
O
O
Cherlet
M
M
Marique
T
T
Renemann
G
G
Loa
A
A
Kretzmer
G
G
Werenne
J
J