대장균에서 증식속도 조절에 의한 수용성 재조합 인간 과립구 콜로니 촉진인자의 생산
Production of Soluble Recombinant Human Granulocyte Colony Stimulating Factor in E. coli by Controlof Growth Rate.
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인간 과립구 성장인자(hG-CSF)는 골수에서 생산되는 단백질로 호중구의 분화 및 생성을 촉진시키는 역할을 한다. 현재 재조합 hG-CSF는 암화학요법에 의한 호중구감소증, 골수이식시 호중구 감소증, 재생불량성 빈혈에 수반되는 호중구 감소증 등으로 적응증이 확대되고 있다. 본 연구에서는 OmpA signal sequence를 삽입하여 인간 과립구 성장인자(hG-CSF)가 분비발현되도록 고안된 T7 promoter에 의하여 발현되는 pYRC1 발현벡터를 제조하였다. E. coli BL21 (pYRC1) 발현시 37oC에서 배양하는 경우 많은 양의 봉입체(aggregates)를 형성한다. 이에 비하여 10 g/l glucose를 포함하는 변형된 MBL 배지에서 10 mM IPTG를 유도물질로 7 시간동안 25oC에서 배양하였을 때 전체 periplasm 단백질의 15%가 soluble rhG-CSF이었다. 또한, 유가식 배양방법을 사용하여 E. coli BL21(pYRC1)에서 soluble rhG-CSF의 생산 조건을 조사하였다. 유가식 배양에서 rhG-CSF의 발현량이 비증식속도를 0.43 h-1에서 0.14 h-1으로, 유도 배양시간을 최적화함으로써 rhG-CSF의 발현량이 4.4 mg/l에서 24 mg/l로 증가하였다.
Abstract
Human granulocyte colony-stimulating factor (hG-CSF) is a hematopoiesis agent that principally affects the differentiation of neutrophils in the bone marrow. At present, recombinant hG-CSF is used successfully in the treatment of chemotheraphy-induced neutropenia and its indication has been expanded to bone marrow transplantation and aplastic anemia. In this study, we have constructed rhG-CSF secretion plasmid pYRC1 in which OmpA signal sequence/hG-CSF gene was expressed under the control of the T7 promoter. rhG-CSF produced in E. coli BL21 (pYRC1) grown at 37oC was found in aggregates. However, 15% of the periplasmic protein was soluble rhG-CSF when the E. coli BL21 (pYRC1) was cultured at 25oC for 7 h in the modified MBL medium containing 10 g/l glucose with 10 mM IPTG induction. The production of soluble rhG-CSF in E. coli BL21 (pYRC1) using fed batch culture was also studied. In the fed batch culture system, the final yield of rhG-CSF produced from E. coli BL21 (pYRC1) was increased from 4.4 mg/l to 24 mg/l by controlling the specific growth rate from 0.43 h-1 to 0.14 h-1, and optimizing the time of induction.
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