(A) study on the human interleukin-6 production by recombinant escherichia coli = 재조합 대장균에 의한 인간 인터루킨-6 생산에 관한 연구

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The major objective of this study was to develop a recombinant E. coli strain, to identify fed-batch culture conditions optimal for the production of human interleukin-6 (hIL-6) in cytoplasm in a soluble form. Five expression vectors were developed for the expression of hIL-6 and hIL-6s fused with NusA, maltose binding protein (MBP), thioredoxin (Trx) or ubiquitin (Ubi). A series of flask cultures were conducted in LB medium at 37℃. The intact hIL-6 was expressed mostly in the form of inclusion body. More than 95% of the hIL-6 fused with NusA (NusA/hIL-6) and about 90% of MBP/hIL-6 were expressed in a soluble form, while Trx/hIL-6 and Ubi/hIL-6 were expressed mostly in the form of inclusion body. Based on this result, NusA was selected the fusion partner for the production of hIL-6 in the subsequent experiments. A series of pH-stat fed-batch cultures of E. coli BL21(DE3) transformed with the NusA/hIL-6 expression vector were conducted in a bioreactor with a working volume of about 3 liters. As the amount of nitrogen source was increased in the feeding medium, the fraction of soluble NusA/hIL-6 increased about 4-folds, while the total amount rather slightly increased. Under the best conditions tested, about 90% of NusA/hIL-6 was produced in the soluble form. The concentration of soluble NusA/hIL-6 was 7.5 g/L with a volumetric productivity of 0.41 g/L-h. The expression of NusA/hIL-6 was confirmed by western blot analysis. The bioefficacy of the NusA/hIL-6 produced was investigated by cell proliferation assay. The hIL-6 activity of NusA/hIL-6 in the cell lysate was 60 ~ 82% of that of the standard hIL-6. A comparative proteome analysis by two-dimensional electrophoresis (2-DE) was carried out to investigate proteins expression patterns in fed-batch cultures. The introduction of NusA was observed to induce several cellular proteins including molecular chaperones such as DnaK, groEL, and trigger factor. The existence of the molecular chaperones was considered to ha...
Advisors
Chang, Yong-Geunresearcher장용근researcher
Description
한국과학기술원 : 생명화학공학과,
Publisher
한국과학기술원
Issue Date
2005
Identifier
245052/325007  / 000995104
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 생명화학공학과, 2005.2 , [ ix, 99 p. ]

Keywords

fed-batch culture; soluble expression; fusion protein; Human interleukin-6 (hIL-6); recombinant E. coli; 재조합 대장균; 유가식 배양; 수용성발현; 융합단백질; 인간인터루킨-6

URI
http://hdl.handle.net/10203/28965
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=245052&flag=dissertation
Appears in Collection
CBE-Theses_Ph.D.(박사논문)
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