재조합 단백질의 효율적 분비 생산을 위한 ABC Transporter시스템의 개발

학위논문(박사) - 한국과학기술원 : 생명과학과, 2007.2, [ vii, 96 p. ]The ABC transporter apparatus of the Gram-negative bacterial type I secretion pathway has been used to secrete many recombinant proteins. The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1 mediated the secretion of a thermostable lipase (TliA). To increase the secretion level of TliA, we firstly optimized the expression level and expression timing of tliA and tliDEF. The four types of co-expression systems for tliA and tliDEF were constructed. When the relative expression levels were changed by adding different concentrations of IPTG, the secretion (16.9 U/ml) of TliA in E. coli [pTliDEFA-223 + pACYC184] was significantly higher than E. coli [pKK223-3 + pTliDEFA-184] secreting the lowest level of TliA (5.2 U/ml). The secretion level of TliA of E. coli [pTliDEFA-223 + pACYC184] was achieved to 26.4 U/ml by inducing gene expression at culture initiation time. Secondly, The ABC transporter (TliDEF) was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency for TliA. TliD mutants with increased secretion efficiency were identified by co-expressing the mutated tliD library with the wild-type tliA in E. coli, and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24 and T35, showed 3.2- (36.4 U/ml), 2.6- (29.2 U/ml), 2.9- (32.8 U/ml) and 3.0-fold (34.0 U/ml) increases of the secretion level of TliA, respectively, but had almost the same expression level of TliD in membrane as that of the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA was mediated by these transporter mutations. Each mutant had a single amino acid change within the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of target protein. Finally, we tried to produce...한국과학기술원 : 생명과학과

Pseudomonas fluorescens SIK W1 ABC transporter를 이용한 단백질 가수 분해 효소 및 외래 단백질의 분비

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