Cellular engineering of escherichia coli for the efficient production of recombinant protein = 재조합 단백질의 고효율 생산을 위한 대장균의 세포공학적 연구

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Escherichia coli, gram negative bacteria, contains two membrane (inner and outer membrane) and can be divided three compartments by these two membrane: cytoplasmic, periplasmic, and extracellular space. The production of recombinant proteins are targeted to one of these three compartments and the mode of gene expression affects the location of the protein produced. In this study, expression systems for the new efficient expression system of recombinant proteins in three compartments of E. coli were developed. Firstly, the efficient expression system into cytoplasmic space of E. coli was developed. Human leptin was produced as inclusion body using T7 promoter in E. coli BL21(DE3). In the fed-batch fermentation with induction at OD=90, 9.7 g/L (37.5 % of total proteins) of leptin was produced. After simple purification steps, 144.9 mg of leptin with a purity of greater than 90% was obtained from the 50 mL culture with a recovery yield of 41.1%. Secondly, the efficient expression system for the secretory production into periplasmic space of E. coli was developed. Human leptin was produced efficiently into the periplasmic space of E. coli using endoxylanase signal peptide. Under Trc promoter, human leptin was secreted successfully into periplasm, and the efficiency of secretion was as high as 95%. However, most of secreted leptin (about 90%) formed the insoluble inclusion body in the periplasm of E. coli. Using periplasmic foldase (DsbA), 69% of secreted leptin was produced as soluble form. Recombinant fusion hG-CSF protein was secreted into the periplasmic space of E. coli using endoxylanase signal peptide. Using modified gene sequence in N-terminal of hG-CSF gene, hG-CSF protein was accumulated up to 48% of total proteins in cytoplasm of E. coli. Fusion of signal peptide directly to hG-CSF protein caused cell lysis after inducion. However, insertion of small peptide (13 a.a.) between the signal peptide and the mature protein allowed the high production of hG-CS...
Advisors
Lee, Snag-Yupresearcher이상엽researcher
Description
한국과학기술원 : 화학공학과,
Publisher
한국과학기술원
Issue Date
2001
Identifier
165741/325007 / 000965352
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 화학공학과, 2001.2, [ viii, 121 p. ]

Keywords

Escherichia coli; 대장균

URI
http://hdl.handle.net/10203/28859
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=165741&flag=dissertation
Appears in Collection
CBE-Theses_Ph.D.(박사논문)
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