(The) secretion of protease and foreign protein by ABC transporter from Pseudomonas fluorescens SIK W1 = Pseudomonas fluorescens SIK W1 ABC transporter를 이용한 단백질 가수 분해 효소 및 외래 단백질의 분비

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Pseudomonas fluorescens is a gram negative psychrotrophic bacterium which secretes a thermostable lipase into the extracellular medium. During the previous study, the lipase of P. fluorescens SIK W1 was found to have a potential C-terminal signal sequence and be secreted by ABC transporter. Genetic loci around the lipase gene were searched and secretory genes were identified. Nucleotide sequencing of a 8.5 kb DNA fragment revealed the three components of the ABC transporter, tliD, tliE, tliF upstream of the lipase gene, tliA. In addition, genes encoding a protease and a protease inhibitor were located upstream of tliDEF. In this study, we examined whether the ABC transporter was responsible for secretion of the protease and could be used for secretion of foreign protein which was not secreted in E. coli. We found that P. fluorescens SIK W1 secreted this protease. When the protease gene (prtA) and ABC transporter genes (tliDEF) were introduced in E. coli, the recombinant strain could also secret the protease. And when both the protease gene and ABC transporter genes were introduced additionally into P. fluorescens SIK W1, the secreted protease was increased by ten fold compared to wild type strain. In addition, when both the C-terminal signal sequences of the protease and lipase were fused to the Proteus vulgaris K80 lipase which was not secreted in E. coli, the fusion lipase had lipase enzyme activity. And E. coli harboring the fusion lipase gene and tliDEF gene secreted the fusion lipase to the culture medium. This results demonstrated that we can secrete the enzyme which was not secreted to culture medium, having enzyme activity, if we use the C-terminal signal sequence and P. fluorescens SIK W1 ABC transporter.
Rhee, Joon-Shick이준식
한국과학기술원 : 생물과학과,
Issue Date
158702/325007 / 000983333

학위논문(석사) - 한국과학기술원 : 생물과학과, 2000.2, [ vii, 70 p. ]


Protease; ABC transporter; Secretion; Foreign protein; 외부 단백질; 단백질 가수분해 효소; ABC 분비시스템; 분비

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