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標題: | 第一部份:酵母菌dehydrodolichyl pyrophosphate synthase的表達及測試A154在控制?物長度的角色 第二部份:鱟血清中脂多醣結合蛋白的活性相關之雙硫鍵位置 PartⅠ: Expression of Yeast Dehydrodolichyl Pyrophosphate Synthase and Examining the Role of A154 in Controlling Product Chain Length PartⅡ: Essential Disulfide Bridge for Lipopolysaccharide Binding Protein from Taiwanese Tachypleus tridentatus |
作者: | Shiao-Cheng Chuang 莊少鈞 |
出版年 : | 2003 |
學位: | 碩士 |
摘要: | 中文摘要 第一部份:大腸桿菌的十一異戊二烯焦磷酸合成醃(UPPs)負責製造胞壁合成所需的長鏈物。此酵素活性區為二個α helices及四個β strands所包圍形成隧道般的中空組成。我們先前的研究已發現有一L137氨基酸位於隧道般的活性區盡頭阻檔C55產物的持續延長因而決定了最後的產物長度?Ko ,T. P., Chen, Y. K., Robinson, H., Tsai, P. C., Gao, Y.-G., Chen, A. P.-C.,Wang, A. H.-J., and Liang, P. H., (2001)J. Biol. Chem. 276, 47474-474821?。將L137定點突變為Ala則產生主要為C70產物。而在和UPPs有sequence homology但主要產物為較長的C75的酵母菌dehydrodolichyl pyrophosphate synthase (DDPPs)上,對應於L137的氨基酸為A154。本論文描述用Pichia pastoris表達yeast的DDPPs及突變A154L來試驗A154在控制DDPPs酵素產物長度的角色。在蛋白表達上我發現使用帶有。Factor signal sequence的質體無法使DDPPs分泌至胞外,而欲用NiNTA純化在X33酵母菌胞內帶有His-tag的重組蛋白卻得到alcohol dehydrogenase但仍能偵測到DDPPs活性。改使用另一表現帶有S-tag重組蛋白的質體配合S-tag親合管柱才有辦法純化到DDPPs(但合成較短的產物)。最復使用KM71菌NiNTA純化後可在SDS-PAGE上看到DDPPs。在無Triton狀態DDPPs主要產生C70(C55:C60:C65:C70:C70:C80=21:17:22:26:12:2),而A154L主要產生較短C55 and C60(C55:C60:C65:C70=31:35:26:8)。當有Triton時酵素主要產生C60(C40:C45:C60:C66=2:45:51:2),而A154L主要產生C55(C40:C45:C50:C55:C60=4:21:28:43:4)。結果顯示A154可能位於產物形成過程的重要樞紐而將其改成較大的Leu導致空間阻礙而使產物變短。 第二部份:鱟血清外源凝集素-1和-2在天賦免疫系統抵禦上,扮演和細菌結合的功能。而外源凝集素-2是一個脂多醣結合蛋白。先前研究顯示外源凝集素-2醣化序列的N3D突變並不影響其功能。然而就蛋白組成而言、分子間雙硫鍵對於鱟血清外源凝集素-2的活性是必需的(郭墩珣碩士論文,2001台大理學院生化所)。本論文將外源凝集素-2上所有的Cys分別置換成Ser來看哪些雙硫鍵的玻壞對脂多醣結合的活性產生影響。我們使用酵母菌來表現這些突變種蛋白,發現C4S的改變使外源凝集素-2失去和脂多醣的結合活性,顯示Cys4可能生成分子間雙硫鍵。和序列相似的TL-3比較,外源凝集素-2多出Cys4可用以形成分子間雙硫鍵。而其他的Cys則可能和TL-3一樣形成分子內雙硫鍵。然而我們卻發現C6S具活性且在non-reducing gel形成monomer。這個突變蛋白可能因形成非共價鍵oligomer而具有活性。使用SephadexG-200 size column,我驗證此C6S可形成hexamer。 ABSTRACT Part I. E. coli undecaprenyl pyrophosphate synthase (UPPs) catalyzes the synthesis of lipid carrier used in making bacterial cell wall. The enzyme active site is surrounded by two α helices and four β strands to form a tunnel-shaped crevice. We have previously reported that the large hydrophobic residue L137 on the bottom of the tunnel provides the seal to block the further chain elongation of the C55 product (Ko, T. P., Chen, Y. K., Robinson, H., Tsai, P. C., Gao, Y-G., Chen, A. P.-C., Wang, A. H.-J., and Liang, P. H., (2001) 1 Biol. Chem. 276, 47474-47482). Substitution of L137 with smaller Ala in UPPs results in formation of longer C70 product. The yeast dehydrodolichyl pyrophosphate synthase (DDPPs) that has sequence homology with UPPs but catalyzes longer C75 product has Ala (A154) in the position corresponding to L137. In order to examine the role of A154 in controlling the product chain length of yeast DDPPs, the wild type and mutant A154L were first expressed using Pichia pastoris X33. I found that N-terminal α-factor signal sequence failed to produce secreted DDPPs and the attempt of purifying recombinant His-tagged DDPPs using NiNTA yielded a major alcohol dehydrogenase band on SDS-PAGE, but the mixture contained DDPPs activity. The construction of expression plasmid containing S-tag allowed the purification of recombinant DDPPs using S-tag affinity column (but the enzyme produced shorter products). Finally by using yeast Pichia pastoris KM71, the DDPPs after NiNTA column chromatography showed a visible band on SDS-PAGE. In the absence of Triton, wild-type DDPPs generated C55-C75 with C70 as major product (C55:C60:C65:C70:C75:C80=21:17:22:26:12:2). In contrast, A154L mainly produced shorter C55 and C60 (C55:C60:C65:C70=31:35:26:8). In the presence of Triton, the wild-type enzyme synthesizes C60 (C50:C55:C60:C66=2:45:51:2), whereas the mutant generates C55 and shorter polymers as final product (C40:C45:C50:C55:C60=4:21:28:43:4). These results suggest that the small A154 is required for DDPPs to synthesize longer products compared to UPPs. PartⅡ: Tachypleus Plasma Lectin-l and-2 (TPL-1 and -2), which bind invading bacteria, play important role in innate host defense. TPL-2 is a bacterial lipopolysaccharide (LPS) binding protein. Previously, we found that N-glycosylation N3D mutant of TPL-2 retains bacterial binding activity but DTT treatment to break disulfide linkages abolishes TPL-2 activity (Kuo Tun-Hsun's master thesis, 2001, National Taiwan University, Graduate Institute of Biochemical Sciences). In this study, all the Cys in TPL-2 was respectively substituted with Ser to examine which Cys is important for TPL-2 activity. Pichia pastoris was utilized to express mutant TPL-2 and the proteins containing α-factor signal sequence were secreted into medium and the active proteins could be easily purified using LPS-Sepharose CL-4B column. Only C4S mutant was found to be inactive, suggesting that the disulfide bond formed via this Cys is essential to form a functional TPL-2. Compared to TL-3 which has sequence homology with TPL-2, there is extra Cys4 in TPL-2. All the Cys in TL-3 have been shown to form interamolecular disulfide bonds and Cys4 in TPL-2 likely forms an intermolecular disulfide linkage. However, we found that the TPL-2 mutant C6S was active but showed a monomer in non-reducing gel. This mutant may form active oligomer through non-covalent interaction. Sephadex G-200 size column was used to display that C6S was indeed a hexamer. |
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