Genetic strategies for production of native-sized spider dragline silk protein in escherichia coli = 대장균에서 자연적 크기의 거미드래그라인실크 단백질 생산을 위한 유전적 전략 연구

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Spider dragline silk is an extremely strong and elastic fiber that makes it attractive for numerous applications. There have been many attempts to make similar high quality fibers by biomimetic spinning of recombinant dragline silk proteins. However, production of native-sized recombinant silk proteins (250-320 kDa) has been unsuccessful because of the innate limitation of Escherichia coli expression system. This study aim to produce native-sized recombinant dragline silk protein with higher titer in E. coli. The 96-repeats dragline silk protein of Golden orb-weaver spider was properly expressed in E. coli cell possessing elevated pool of glycyl-tRNA and co-overexpressed factor for inversion stimulation (Fis) which encodes the transcriptional activator of ribosomal RNA operons. Co-overexpression of Fis was assumed to enhance the expression level of 96-repeats Golden orb-weaver spider dragline silk protein by the mechanism of up-regulation of the ribosomal pool and several translation related protein in the nutritious environment. For the production of 96-repeats Black widow spider dragline silk protein, the host strain E. coli BLR strain was employed to minimize homologous recombination of recombinant gene. When RraA encoding RNase E inhibitor protein was co-overexpressed with Fis, the expression level of 96-repeats Black widow spider dragline silk protein was dramatically enhanced. Finally, fed-batch fermentation of E. coli BLR strain which engineered to co-overexpress Fis and RraA, resulted in $0.7 g l^{-1}$ of the 96-repeats Black widow spider dragline silk protein at the 10 hour after induction. These results provide insight into general genetic strategies for expression of proteins with high molecular weight and repetitive genetic structure to achieve higher production in E. coli.
Advisors
Lee, Sang Yupresearcher이상엽researcher
Description
한국과학기술원 :생명화학공학과,
Publisher
한국과학기술원
Issue Date
2011
Identifier
325007
Language
eng
Description

학위논문(석사) - 한국과학기술원 : 생명화학공학과, 2011.8 ,[vi, 47 p. :]

Keywords

Escherichia coli; spider dragline silk; BLR(DE3); Fis; RraA; 대장균; 거미드래그라인실크

URI
http://hdl.handle.net/10203/221510
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=656999&flag=dissertation
Appears in Collection
CBE-Theses_Master(석사논문)
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