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dc.description.abstractYeast S. cerevisiae has long been GRAS organism and used as a host for the production of human therapeutic proteins. Although it has many advantages for the production of human proteins, the secretion level varies considerably according to the type of proteins. To minimize the inconsistency between proteins and improve the secretion level of “difficult-to-express” proteins, we, previously, developed a high throughput screening system for the optimal translational fusion partner (TFP) by an invertase-based autoselection system. To screen an optimal TFP for the secretory production of a novel human cytokine IL-32 which is an inducer of TNFα, IL-32α, IL-32β and IL-32γ gene were inserted into vectors containing 5x103 TFP library and invertase gene (SUC2) via in vivo recombination. For IL-32α, over two hundred transformants and IL-32β, 16 transformants and IL-32γ, 11 transformants appeared on a medium containing sucrose as a sole carbon source. Removal of SUC2 and insertion of IL-32termination codon into the vector library isolated from all transformants were performed by PCR, simultaneously. After retransformation of PCR product and vector fragment into yeast, for IL-32α, randomly selected 40 transformants were tested for their secretion of IL-32 in culture supernatant. Five different TFPs were simply obtained for the optimal secretion of IL-32. Among them, TFP3 was selected for the optimal secretion of IL-32α. Fed batch fermentation of selected strain revealed the secretion of IL-32a up to 300 mg/liter into culture supernatant. Three different TFPs were obtained for IL-32β and 2 different TFPs were obtained for IL-32γ. Selected TFPs could also improve the secretion level of IL-32β and IL-32γ, respectively. Each recombinant strain developed in this study will be useful for the production of three novel cytokines, IL-32α, β and γ.-
dc.description.tableofcontentsI. 서론 = 1 A. 재조합 단백질 발현 시스템 = 1 B. 맞춤형 분비 융합 인자 기술 (TFP technology) = 9 C. 신규 인체 cytokine Interleukin-32 = 12 II. 실험 재료 및 방법 = 15 A. 실험 재료 = 15 B. 실험 방법 = 20 1. IL32-α, IL32-β 및 IL32-γ 유전자의 확보 = 20 2. E.coli transformation 과 plasmid DNA 분리 = 20 3. Yeast transformation = 22 4. 효모로부터 total DNA 의 분리 = 23 5. SDS-PAGE 과 Western blot analysis = 24 6. Fermentation = 27 III. 결과 = 29 A. IL-32 의 TFP 발굴 library 제작 = 29 1. In vivo recombination 을 통해 TFP library에 IL-32 유전자 도입 = 29 2. Autoselection system을 이용하여 library screening = 33 B. TFP library 를 이용 IL-32α의 발현에 적합한 TFP 발굴 = 33 1. IL-32α의 발현 vector 제작 및 발현 확인 = 33 2. TFP 선별과 sequencing = 39 3. UTR (5’-untranslated region) 제거 효과 = 41 4. IL32 polyclonal antibody 와 HIS antibody 비교 = 47 5. TFP 3 변이체를 이용한 IL-32α 분비량 비교 = 49 6. 유가식 발효 (Fed-batch fermentation)을 이용한 IL-32α 의 분비생산 = 53 C. IL-32β와 IL-32γ 의 분비생산에 적합한 TFP 발굴 = 56 1. TFP library 로부터 IL-32β 와 IL-32γ 의 발현에 적합한 TFP 클론 획득 = 56 2. TFP의 확인 및 TFP를 이용한 IL-32β 와 IL-32γ의 발현 확인 = 57 3. UTR 유무에 따른 IL32 - β 와 IL32 - γ의 발현 차이 비교 = 60 4. 최종 선별 균주의 유가식 발효배양(fed-batch fermentation)을 통한 IL-32β 및 IL-32γ의 생산 = 62 IV. 고찰 = 68 V. 참고 문헌 = 71 VI. Abstract = 78-
dc.format.extent1439224 bytes-
dc.publisher이화여자대학교 대학원-
dc.title효모 Saccharomyces cerevisiae의 맞춤형 분비융합인자 기술을 이용한 인체 interleukin-32의 분비생산 연구-
dc.typeMaster's Thesis-
dc.format.pagev, 79 p.-
dc.identifier.major대학원 생명과학과- 2-
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