Proteomic analysis of escherichia coli and its application for recombinant proteins production = 대장균 균주의 단백질체 분석과 재조합 단백질 생산을 위한 그 응용

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First, the extracellular proteome of E. coli BL21(DE3) in high cell density cultivation was firstly profiled and compared with that of E. coli W3110. These data could be useful for the study of excretion mechanism of nonpathogenic E. coli and applied for the fusion target identification in extracellular recombinant proteins production. Second, based on the above study, a new system was established for high-level extracellular production of recombinant proteins in E. coli. The best excreting fusion partner, OsmY, was identified and used as a carrier protein to excrete heterologous proteins into the culture medium. Model proteins including E. coli alkaline phosphatase, Bacillus subtilis α-amylase, and human leptin could all be excreted into the medium at concentrations ranging from 5 to 64 mg/L during the flask cultivation. Third, based on comparative proteomic analysis, a platform was developed for high-level cytoplasmic production of spider silk proteins in E. coli. To get a global view of the responses upon silk protein production, the whole proteomes of silk protein-producing cells were profiled. The comparative proteomic analysis implied that intracellular glycyl-tRNA pool probably limited the production of the silk proteins. Therefore, measures were taken to increase glycine tRNA levels by coexpression of the glycine tRNA genes (glyVXY), and increase glycine availability by coexpression of the glycine biosynthetic gene (glyA) and inactivation of the glycine cleavage system. Severalfold increase was thus achieved in the production of the silk proteins, ranging from ~100 to 300 kDa. These findings may be potentially useful for the large-scale production of recombinant proteins in E. coli.
Lee, Sang-Yupresearcher이상엽
한국과학기술원 : 생명화학공학과,
Issue Date
586359/325007  / 020054512

학위논문(박사) - 한국과학기술원 : 생명화학공학과, 2009.2, [ x, 95 p. ]


Escherichia coli; 견사단백질; 비교단백체학; 단백질 생산; 세포외 단백체; 대장균; Extracellular proteome; protein production; comparative proteomic; silk protein

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