重組次單位巴斯德桿菌毒素在大腸桿菌中之表現

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標題:	重組次單位巴斯德桿菌毒素在大腸桿菌中之表現 Expression of Recombinant Subunit Protein of Pasteurella multocida toxin in Escherichia coli
作者:	林怡妏 Yi-wen Lin
貢獻者:	吳明娟邱淑媛嘉南藥理科技大學：生物科技研究所
關鍵字:	豬萎縮性鼻炎巴斯德桿菌毒素高密度培養醱酵槽大腸桿菌 Progressive atrophic rhinitis (PAR) Pasteurella multocida toxin (PMT) Escherichia coli Fermentor High cell density culture (HCDC)
日期:	2008
上傳時間:	2009-03-11 11:41:58 (UTC+8)
摘要:	本研究探討攜帶有豬萎縮性鼻炎次單位毒素Tox1基因之重組大腸桿菌大量表現重組蛋白質的最適條件，並對Tox1進行部份純化。第一部分為搖瓶試驗，試驗溫度、pH值、誘導時間、誘導劑濃度等因子在LB培養基中對蛋白質表現之影響。結果顯示IPTG濃度不影響誘導效果，最適誘導時間為接種後3~4小時，最適培養pH值介於6.16~7.87，於25~37oC之間誘導效果相當，Tox1產量約為0.1 g/L。第二部分為醱酵槽培養，分別以SSP、modified R及高密度培養基利用DO-stat醱酵策略探討重組蛋白質表現之最適條件。結果顯示重組大腸桿菌利用SSP培養基批式培養並以IPTG誘導後，菌體濃度（OD600）可達15，Tox1最大產量為0.5 g/L，單位時間產率可達50~60 mg/L/h，在modified R培養基批式培養中，菌體濃度可達53，Tox1產量為1.5 g/L，單位時間產率可達160 mg/L/h。在高密度饋料培養條件下，菌體濃度（OD600）可達180，Tox1最大產量為5.8 g/L，單位時間產率可達370 mg/L/h。第三部分Tox1之性質及純化，發現以高密度培養並於30oC誘導之菌體，有74%之Tox1以可溶性狀態存在，且只有28%的可溶性Tox1可利用10~35%硫酸銨沉澱。Tox1在純化過程中會逐漸降解，成為74 kDa與66 kDa兩個分子量，因此無法測得可溶性Tox1之等電點。 In this study, the effects of culture conditions on the growth of recombinant E. coli BL21 (DE3) and the expression of subunit protein of Pasteurella multocida toxin 1 (Tox1) were investigated in both shaking flask and fermentor. In shaking flask, the optimal condition for the expression of Tox1 was by culturing E. coli in LB medium at 37oC with initial pH 6.17-7.87 until mid-log phase, which is about 3-4 h after inoculation. Inducer, IPTG (2.5~400 M), was then administrated and cells were cultured at 25-37oC for another 3- 7 h and the yield of Tox1 could be as high as 0.1 g/L. Three different media, i.e. SSP, modified R and high density culture (HCDC), were used to study the optimal condition in fermentor in dissolved oxygen-stat (DO-stat) culture. It was found that cell density of 15 OD600 units and 0.5 g/L Tox1 (50-60 mg/L/h) could be obtained when recombinant E. coli was cultured in SSP medium at 37oC until mid-log phase followed by induction and culture with IPTG (100-200M) at 25oC. Higher cell density of 53 OD600 units and 1.5 g/L Tox1 (169 mg/L/h) could further be achieved by changing to modified R culture with similar culture/induction parameters. The highest yield which was cell density of 180 OD600 units and 5.8 g/L Tox1 (370 mg/L/h), was obtained when recombinant E. coli was cultured in HCDC with feeding. In contradict to previous literature, the majority of recombinant Tox1 (74%) was expressed in soluble form inside E. coli. Only 28% of the soluble Tox1 could be purified by precipitation with 10-30% ammonium sulfate. The purified Tox1, however, was degraded into 74 kDa and 66 kDa fragments so that no exact PI could be determined.
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