High level secretory production of hG-CSF by fed-batch culture of recombinant escherichi coli = 재조합 대장균의 유가식 배양을 이용한 hG-CSF의 고농도 분비생산

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High level secretory production of human granulocyte-colony stimulating factor (hG-CSF) by fed-batch cultures of Escherichia coli MC4100 harboring plasmid pTHKCSFmII was investigated in both lab-s cale and 30L pilot-scale fermentors. The best medium and the optimal initial glucose concentration for the production of hG-CSF and cell growth were determined to be modified R medium and 20g/L by flask cultures. And the IPTG (isopropyl-β-D-thiogalactopyranoside, MW=238.3) concentration at a 1mM is enough to induce the expression of hG-CSF gene. The effect of induction time on hG-CSF protein production by batch and fed-batch culture of recombinant E. coli was examined. Induction at higher cell density (late-exponential phase) was more efficient than induction at lower cell density (early-exponential phase and mid-exponential phase) for the hG-CSF protein production. Among three different feeding strategies (pH-stat, exponential, and constant feeding), the constant (0.228 g glucose/min) and exponential feeding strategies (specific growth rate of 0.116) were better than the pH-stat for hG-CSF production. Therefore, an optimal condition for hG-CSF production was to induce at higher cell density phase using constant (0.228 g glucose/min) or exponential (specific growth rate of $0.116h^{-1}$) as a feeding strategy. Under these conditions, 4.35 g/L of hG-CSF protein could be produced in a secretory form. The same feeding strategy that resulted in best performance in lab-scale fermentation was applied in pilot-scale 30 L fermentor. The scale-up strategy of constant dissolved oxygen level was used. The dissolved oxygen level was maintained above 30% of air saturation by supplying air (3 vvm) and by automatically controlling the agitation speed up to 500 rpm. The maximum dry cell weight and concentration of hG-CSF were 51.74 g/L and 4.23 g/L, respectively. These results were similar with the lab-scale fermentation, so the scale-up study in a pilot-scale 30 L fermentor was su...
Chang, Ho-Namresearcher장호남researcher
한국과학기술원 : 화학공학과,
Issue Date
165629/325007 / 000993448

학위논문(석사) - 한국과학기술원 : 화학공학과, 2001.2, [ viii, 52 p. ]


scale-up; secretion; fed-batch; hG-CSF; High level production; 대량 생산; 분비생산; 스케일업; 유가식 배양; 인체유래 G-CSF

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