Development of gene expression systems and high cell density cultivation for the production of bioadhesive precursor protein = 생물접착제 발현을 위한 유전자 발현 시스템의 개발 및 고농도 배양

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In nature when materials need to be joined in most cases the only option is via adhesion, often promoted by adhesives. Since bioadhesives of marine organisms (such as mussels, barnacles and oysters) are applied and cured in moist environments, they are potentially useful for many medical and dental applications. Mussel adhesive protein is comprised mainly of about 75 repeats of the decapeptide sequence Ala-Lys-Pro-Ser-Tyr-HydroxyPro-HydroxyPro-Thr-Dopa-Lys. Biological synthesis system of bioadhesive protein analog in Escherichia coli was developed, because isolation of the uncured adhesive from mussels for commercial use is not practical. For the expression of bioadhesive precursor protein in a tandem repeats, 4 expression vectors using inducible T7 expression system were developed and successfully expressed the bioadhesive precursor protein with various molecular weight. Orientation of the repeats was suppressed by the complementary nature of nonpalindromic ends generated in BlpI. High cell density culture and high level expression are essential for the mass production of bioadhesive precursor protein. In order to achieve these goals, the effects of T7 lysozyme and plasmid size on the cell growth and production of a bioadhesive precursor protein were studied in fed-batch culture. Cell growth and bioadhesive precursor protein expression level were not significantly different between BLR(DE3)[pKYC0.8] and BLR(DE3)[pLysS] [pKYC0.8] in batch culture. In fed-batch culture, however, BLR(DE3) [pLysS][pKYC0.8] showed growth retardation and much lower level of bioadhesive precursor protein expression than that obtained with BLR(DE3)[pKYC0.8]. This result suggests that the presence of pLysS in the T7 expression system strongly affects the cell growth and expression of BP protein in high cell density cultivation. Therefore, if the target protein is not toxic to the cell, higher expression level of foreign protein can be obtained with BLR(DE3) strain without pLysS in hi...
Advisors
Chang, Ho-Namresearcher장호남researcher
Description
한국과학기술원 : 화학공학과,
Publisher
한국과학기술원
Issue Date
1998
Identifier
143516/325007 / 000935806
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 화학공학과, 1998.8, [ xiii, 105 p. ]

Keywords

Fed-batch culture; High cell density culture; T7 expression system; Bioadhesive; T7 lysozyme; T7 라이소자임; 유가식 배양; 고농도배양; T7 발현 시스템; 생물접착제

URI
http://hdl.handle.net/10203/28781
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=143516&flag=dissertation
Appears in Collection
CBE-Theses_Ph.D.(박사논문)
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