請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75147
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.author | Jia-Yuan Chen | en |
dc.contributor.author | 陳嘉元(冠廷) | zh_TW |
dc.date.accessioned | 2021-07-01T08:11:59Z | - |
dc.date.available | 2021-07-01T08:11:59Z | - |
dc.date.issued | 2000 | |
dc.identifier.citation | (1) Comb, D. G., and Roseman, S. (1960) The sialic acids I. The structure and enzymatic synthesis of N-acetylneuraminic acid. J. Biol. Chem. 235: 2529-2537. (2) Moxon, E. R., and Kroll, J. S. (1990) The role of bacterial polysaccharide capsules as virulence factors. Curr. Top. Microbiol. Immunol. 150: 65-85. (3) Orskov, F., and Orskov, I. (1992) Escherichia coli sertyping and disease in man and animials. Can. J. Microbiol. 38: 699-704. (4) Kaijser, B., Hanson, L. A., Jodal, V., Lindin-Janson, G., and Robbins, J. B. (1977) Frequency of E.coli K antigens in urinary tract infections in children. Lancet. I: 664-666. (5) Bliss, J. M., and Silver, R. P. (1996) Coating the surface: a model for expression of capsular polysialic acid in Escherichia coil K1. Mol. Microbiol. 21 (2): 221-231. (6) Gotschlich, E. C., Fraser, B. A., Nishimura, O., Robbins, J. B., and Liu, T. Y. (1981) Lipid on capsular polysaccharides of Gram- negative bacteria. J. Biol. Chem. 256: 8915-8921. (7) Silver, R. P., Finn, C. W., Vann, W. F., Aaronson, W., Schneerson, R., Kretschmer, P. J., and Garon, C. F. (1981) Molecular cloning of the K1 capsular polysaccharide genes of E. coli. Nature 289: 696-698. (8) Boulnois, G. J., Roberts, I. S., Hodge, R., Hardy, K. B., and Timmis, K. N. (1987) Analysis of the K1 capsule biosynthesis genes of escherichia coli: definition of three functional regions for capsule production. Mol. Gen. Genet. 208: 242-246. (9) Roberts, I. S. (1995) Bacterial polysaccharides in sickness and in health. Microbiology 141: 2023-2031. (10) Vimr, E. R., Steenbergen, S. M., and Cieslewicz, M. (1995) Biosynthesis of the polysialic acid capsule in Escherichia coli K1. J. Industr. Microbiol. 15(4): 352-60. (11) Kr?ncke, K. D., Boulnois, G., Roberts, I., Bitter-Supermann, D., Golecki, J. R., Jann, B., and Jann, K. (1990) Expression of the Escherichia coli K5 capsular antigen: immunoelectron microscopic and biochemical studies with recombinant E. coli. J. Bacteriol.172: 1085-1091. (12) Pavelka, Jr, M. S., Wright, L. F., and Silver, R. P. (1994) Characterization of kpsT, the ATP-binding component of the ABC-transporter involved with the export of capsular polysialic acid in Escherichia coli K1. J. Biol. Chem. 269: 20149-20158. (13) Wunder, D. E., Aaronson, W., Hayes, S. F., Bliss, J. M., and Silver, R. P. (1994) Nucleotide sequence and mutational analysis of the gene encoding kpsD, a periplasmic protein involved in transport of polysialic acid in Escherichia coli K1. J. Bacteriol. 176: 4025-4033. (14) Frosch, M., Edwards, U., Bousset, K., Kraube, B., and Weisgerber, C. (1991) Evidence for a common molecular origin of the capsule gene loci in Gram-negative bacteria expressing group Ⅱ capsular polysaccharides. Mol. Microbiol. 5: 1251-1263. (15) Hashimoto, Y., Li, N., Yokoyama, H., and Ezaki, T. (1993) Complete nucleotide sequence and molecular characterization of viaB region encoding Vi antigen in Salmonella typhi. J. Bacteriol.175: 4456-4465. (16) Kroll, J. S., Loynds, B., Brophy, L. N., and Moxon, E. R. (1990) The bex locus in encapsulated Haemophilus influenzae: a chromosomal region involved in capsule polysaccharide export. Mol. Microbiol. 4: 1853-1862. (17) Troy, Ⅱ, F. A. (1992) Polysialylation: from bacteria to brains. Glycobiology, 2: 5-23. (18) Annunziato, P. W., Wright, L. F., Vann, W. F., and Silver, R. P. (1995) Nucleotide sequence and genetic analysis of the neuD and neuB genes in region 2 of the polysialic acid gene cluster of Escherichia coli K1. J. Bacteriol. 177(2): 312-319. (19) Steenbergen, S. M., and Vimr, E. R. (1990) Mechanism of polysialic acid chain elongation in Escherichia coli K1. Mol. Microbiol. 4: 603-611. (20) Vimr, E. R., Aaronson, W., and Silver, R.P. (1989) Genetic analysis Biosynthesis of chromosomal mutations in the polysialic acid gene cluster of Escherichia coli K1. J. Bacteriol. 171: 1106-1117. (21) Vimr, E. R. (1992) Selective synthesis and labeling of the polysialic acid capsule in Escherichia coil K1 strain with mutations in nanA and neuB. J. Bacteriol. 174(19): 6192-6197. (22) Bliss, J. M., and Silver, R. P. (1996) Coating the surface: a model for expression of capsular polysialic acid in Escherichia coil K1. Mol. Microbiol. 21 (2): 221-231. (23) Rodriguez-Aparicio, L. B., Ferrero, M. A., and Reglero, A. (1995) N-Acetyl-D-neuraminic Acid synthesis in Escherichia coil K1 occurs through condensation of N-Acetyl-D-mannosamine and pyruvate. Biochem. J. 308:501-505. (24) Ferrero, M. A., Reglero, A., Fernandez-Lopez, M., Ordas, R., and Rodriguez-Aparicio, L. B. (1996) N-acetyl-D-neuraminic acid lyase generates the sialic acid for colominic acid biosynthesis in Escherichia coil K1. Biochem. J. 317: 157-165. (25) Watson, D. R., Jourdian, G. W., and Roseman, S. (1966) The sialic acid Ⅷ: sialic acid 9-phosphate synthetase. J. Biol. Chem. 174, 315-319. (26) Blacklow, R. S., and Warren, L. (1962) Biosynthesis of sialic acids by Neisseria meningitides. J. Biol. Chem. 237, 3520-3526. (27) Lzard, T., Lawrence, M. C., Malby, R. L., Lilley, G. G., and Colman, P. M. (1994) The three-dimensional structure of N-acetylneuraminase lyase from Escherichia coli. Structure, 2: 361-369. (28) Vimr, E. R., and Troy, F. A. (1985) Regulation of sialic acid metabolism in Escherichia coli: role of N-acylneuraminate pyruvate-lyase. J. Bacteriol. 164: 854-860. (29) Vimr, E. R., and Troy, F. A. (1985) Identification of an inducible catabolic system for sialic acid (nan) in Escherichia coil. J. Bacteriol. 164: 845-853. (30) Vann, W. F., Tavarez, J. J., Crowley, J., Vimr, E. R., and Silver, R. P. (1997) Purification and characterization of the Escherichia coli K1 neuB gene product N-actylneuraminic acid synthetase. Glycobiology 7 (5): 697-701. (31) Merker, R. I., and Troy, F. A. (1990) Biosynthesis of the polysialic acid capsule in Escherichia coli K1. Cold inactivation of sialic acid synthase regulates capsule expression below 20℃. Glycobiology. 1 (1): 93-100. (32) Komaki, E., Ohta, Y., and Tsukada, Y. (1997) Purification and characterization of N-acetylneuraminate synthase from Escherichia coli K1-M12. Biosci. Biotechno. Biochem. 61 (12): 2046-50. (33) Linton, D., Karlyshev, A. V., Hitchen, P. G., Morris, H. R., Dell A., Gregson, A., and Wren, B. W. (2000) Multiple N-Acetylneuraminic acid synthetase (neuB) genes in Campylobacter jejuni: identification and characterization of the gene involved in sialylation of lipo-oligosaccharide. Mol. Microbiol. 25 (5): 120-1134. (34) Lawrence, S. M., Huddleston, K. A., Pitts, L. R., Nguyen, N., Lee, Y. C., Vann, W. F., Coleman, T. A., Betenbaugh, M. J. (2000) Cloning and Expression of the Human N- Acetylneuraminic Acid (Neu5Ac) Phosphate Synthase Gene with 2-Keto-3-deoxy-D-glycero-D- galacto-nononic Acid (KDN) Biosynthetic Ability. J. Biol. Chem. 275(23): 17869-17877. (35) Nakata, D., Close, B. E., Colley, K. J., Matsuda, T., Kitajima, K. (2000) Molecular Cloning and Expression of the Mouse N-Acetylneuraminic Acid 9-Phosphate Synthase Which Does Not Have Deaminoneuraminic Acid (KDN) 9-Phosphate Synthase Activity. Biochem Biophys Res Commun. 273(2): 642-648. (36) Liu, J. L., Shen, G. J., Ichikawa, Y., Rutan, J. F., Zapata, G., Vann, W. F., and Wong, C. H. (1992) Overproduction of CMP-Sialic Acid Synthetase for Organic Synthesis. J. Am Chem. Soc. 114: 3901-3910. (37) Seed, B., Parker, R. C., and N. Davidson. (1982) Representation of DNA sequences in recombinant DNA libraries prepared by restriction enzyme partial digestion. Gene 19:201-209 (38) Birnboim, H. C. (1983) A rapid alkaline extraction method for the isolation of plasmid DNA. Methods of Enzymology. 100: 243-255. (39) McDonell, M. W., Simon, M. N., and Studier, F. W. (1977) Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J. Mol. Biol. 110: 119-146 (40) Saiki, R. K. and Gelfand, D. H. (1998). Primer-directed Enzymatic amplification of DNA with thermostable DNA polymerase. Science 239: 487-491. (41) Chang, Wei Chao, Chen, J. Y., Chang, T., Liu, M. Y., Payne, W. J., LeGall, J., Chang, W. C. (1998) The C-terminal segment is essential for maintaining the quaternary structure and enzyme activity of the nitric oxide forming nitrite reductase from Achromobacter cycloclastes. Biochem. Biophys. Res. Commun. 250(3): 782-785. (42) Nishimura, A., Morita, M., Nishimura, Y., and Sugino, Y. (1990). A rapid and highly efficient method for preparation of competent Escherichia coli cells. Nucl. Acids Res. 18: 6169 (43) Bujard, H., Gentz, R., Lanzer, M., Stuber, D., Muller, M., Ibrahimi, I., Hauptie, M. T., and Dobberstein, B. (1987) A T5 promotor based transcription-translation system for the analysis of proteins in vivo and in vitro. Methods Enzymol. 155: 416-433. (44) Bush, G. L., Tassin, A., Friden, H., and Meyer, D. I. (1991) Purification of a translocation-competent secretory protein precursor using nickel ion affinity chromatograghy, J. Biol. Chem. 266: 13811-13814. (45) Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 7(72): 248-254. (46) Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, B. K., Goeke, N. M., Olson, B. J., Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150(1): 76-85. (47) Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T., (1995) How to measure and predict the molar absorption cofficient of a protein. Protein Sci. 4: 2411-2423. (48) Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. (49) Schagger, H. and Jagow, G. V. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1-100 kDa. Anal. Biochem. 166: 368-379. (50) Bumette, W. H. (1981) Western Blotting: Electrophoretic transfer of proteins from SDS- polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Ana. Biochem. 112: 195-203 (51) Gracia, E., and Fernandez-Belda, F. (1992) Ponceau S as a dye for quantitative protein assay. Its use in the presence of Triton X-100. Biochem. Int. 27(4): 725-33 (52) Barattl, J., Maroux, S., and Louvard, D. (1973) Effect of ionic strength and calcium ions on the activation of trypsinogen by enterokinase. Biochim. Biophys. Acta. 321: 632-638. (53) Unger, F. M. (1981) The Chemistry and biological significance of 3-deoxy-D-manno-2- octulosonic acid (KDO). Adv. Carbohydr. Chem. Biochem. 38: 326. (54) Skoza, L., and Mohos, S. (1976) Stable Thiobarbituric Acid Chromophore with Dimethyl Sulphoxide. Biochem. J. 159: 457-462 (55) Schauer, R. (1982) Chemistry, metabolism, and biological functions of sialic acids. Adv. Carbohydr. Chem. Biochem. 40: 152-163. (56) Uchida, Y., Tsukada, Y., and Sugimori, T. (1977) Distribution of neuraminidase in Arthrobacter and its purification by affinity chromatography. J. Biochem. 82: 1425-1433. (57) Schauer, R. (1978) Characterization of sialic acids. Methods Enzymol. 50:64-89. (58) Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33(1): 103-119. (59) Farabaugh, P. J. Sequence of the lad gene. Nature (1978) 274(5673): 765-9. (60) Gaboriaud, V., Bissery, V., Benchetrit, T., and Mornon, J. P., (1987) Hydrophobic cluster analysis: an efficient new way to compare and analysis amino acid sequences. FEBS Lett. 224(1): 149-155. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75147 | - |
dc.description.abstract | 位在細菌表面的菌鞘,屬於Kl抗原的α-2,8醣聚合體,扮演著細菌入侵人類或者其他動物體內時,決定對於宿主是否具有毒性的關鍵角色。這些多聚醣的合成,一般都是經由下列步驟所產生:唾液酸的合成、唾液酸經活化形成CMP-唾液酸以及運送唾液酸給適當的受器。目前已有兩種格蘭氏陰性菌唾液酸合成的酵素被研究出來,一種為唾液酸分解?,能夠進行可逆反應,以N-乙醯甘露糖胺酸和丙酮酸進行縮合反應得到唾液酸,一般情況下為代謝分解作用;另一種為唾液酸合成?,為一個不可逆反應,以N-乙醯甘露糖胺酸和磷酸烯醇丙酮酸進行縮合反應得到唾液酸。
本篇研究是利用分生技術選殖大腸桿菌Kl唾液酸合成?,進行大量表現和純化,也對其酵素特性作一番探討。雖然這個酵素在早先已經被表現純化出來,但是其活性非常地低,以致於必需使用缺乏唾液酸分解?的菌種,並加入磷解?抑制劑來改善唾液酸合成?的表現及活性;另外也得以C14同位素標定N-乙醯甘露糖才能檢測唾液酸的產生。在大量表現該蛋白質之後,我們應用Ni2+金屬離子親和性管柱層析法,高度純化N端帶有組氨酸標識之唾液酸合成?,得到較以前人所表現唾液酸合成?高的比活性,並藉由Enterokinase蛋白質水解?將不必要的N端胺基酸序列切除,得到完整的野生型唾液酸合成?。另一方面,我們發現這個酵素是以雙體的形式存在,並含有兩種不同分子量40 kDa和33 kDa單體存在,利用不同的陰離子交換樹脂和逆向層析法,並不能夠將其分離出。現階段我們無法得知為何會有33 kDa單體存在,前人也曾經觀察過這種現象,不過可以確定的是,33 kDa單體是從40 kDa單體上胺基酸K279及I280之間斷裂所得到,而且這種蛋白質水解現象在經過純化以後仍然可以觀察到。 唾液酸合成?最佳酸鹼值在8.5,而在7.0?9.0範圍有較穩定的特性;在溫度45℃下反應性最高,並需要二價 Mg2+或Mn2+離子參與才能有明顯的活性。另外我們以不同醣分子作為唾液酸合成?的受質,發現必須要以甘露糖胺酸為骨架和 N-取代基存在才能有反應性,而其他的醣分子均無反應,因此可能酵素活化區和結合部位與這兩者有密切的關係。而其中N-Acetylglycolyl-D-mannosamine (ManNGcAc)利用Periodate-Thiobarbituric Acid Assay、薄膜層析法以及核磁共振光譜,可以檢測到有相對於N-乙醯甘露糖胺酸的高活性存在;因此可以應用在合成唾液酸衍生物上,但是其低活性的特徵仍是必須克服的障礙。 這是第一篇有關大腸桿菌Kl唾液酸合成?,不需要藉由缺乏唾液酸分解?菌種的表現,就能夠拿到大量且比活性高的報導;另一方面,也觀察到44 kDa之唾液酸合成?會水解產生33 kDa之單體;及利用生物資訊(Bio-Infomatics)分析方式,與其他已知相關唾液酸合成的蛋白質序列比對的結果,推測到兩段具有高度保留性的胺基酸序列:GxSDHxxxxxxxxxAVxxGxxxIEKHF和xxxxxIxAExxxNHNGS,這兩個區域具有疏水性的特徵,以及特定酸鹼催化的胺基酸存在,因此可能在酵素活化區和受質結合區上扮演了重要的角色,這都有待日後作進一步的研究加以證實。 | zh_TW |
dc.description.abstract | The K1 capsular polysaccharide, an α-2,8 linked polymer of N-acetyl-D-neuraminic acid (NeuAc), is an important virulence determinant of extra intestinal neuropathogenic Escherichia coil. The biosynthesis of this sugar polymer is composed of the following steps; synthesis of NeuAc, activation of NeuAC to CMP-NeuAc, and transfer of NeuAc to the acceptor. Two enzymes have been reported for the synthesis of NeuAc in Gram-negative bacteria. One is sialic acid aldolase that catalyzes the reversible reaction from N-acetylmannosamine (ManNAc) and pyruvate to NeuAc. The reaction equilibrium favors the catabolic direction. The other is sialic acid synthetase responsible for the aldol reaction of ManNAc and phospho(enol)pyruvate (PEP) to form NeuAc.
In this thesis, the overexpression, purification and characterization of sialic acid synthetase from E.coil K1 are investigated. Although this enzyme has been cloned and expressed, its activity is so low that past studies had to use sialic acid lyase-deficient mutant strain in a combination with a phosphatase inhibitor to improve the activity of overexpressed synthetase. Additionally, the isotope labeled C14-ManNAc was required to detect the NeuAc activity. The recombinant N-terminal His-tagged sialic acid synthetase, purified by the Ni2+ affinity chromatography, was measured to have higher specific activity than that had been reported previously. The removal of the N-terminal His-tag by the enterokinase digestion led to isolation of the native sialic acid synthetase. This enzyme was determined to exist in heterogeneous dimeric forms. The two subunits of 40 kDa and 33 kDa to constitute these dimers were found difficult to be separated by anion exchange and reverse phase HPLC chromatography. The 33 kDa protein resulted from the cleavage of the 40 kDa protein at K279 of the C-terminal segment. The protein degradation was also observed after purification, similar to the reported previously. The enzyme is stable in the pH range of 7 to 9 with an optimum at 8.5 and thermostable up to 45 ℃. The metal ion, Mg2+ or Mn2+, is required for enzyme activity. Various monosaccharides have been tested for the substrate specificity. The result suggested that the N-substituent at the C-2 position and the D-manno configuration are both important for enzyme specificity. Among the different sugars tested, N-Acetylglycolyl-D-mannosamine (ManNGcAc) has been found to have 85% activity compared to ManNAc, as determined by periodate-thiobarbituric acid assay, thin layer chromatography, and 1H NMR. Therefore, it is possible to synthesize sialic acid derivates by this enzyme but the low reactivity remains as a problem to be overcome. This is the first report for the overproduction of E.coli sialic acid synthetase with high purity over 95% and good activity without using sialic acid lyase-deficient mutant. Finally, the protein sequence analysis suggests that two highly conserved amino acid sequences are rather hydrophobic, GxSDHxxxxxxxxxAVxxGxxxIEKHF and xxxxxIxAExxxNHNGS. They may be involved in either substrate binding or catalysis. | en |
dc.description.provenance | Made available in DSpace on 2021-07-01T08:11:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2000 | en |
dc.description.tableofcontents | 誌謝………………………………………………………………………………………………壹 縮寫表.............................................................................................................................................Ⅰ 中文摘要.............................................................................................................................................Ⅲ 英文摘要.............................................................................................................................................Ⅴ 第一章 緒論 1.1. 大腸桿菌之菌鞘多醣體聚合物....................................................................................................l 1.2. 唾液酸之新陳代謝....................................................................................................4 1.3. 實驗研究概況....................................................................................................10 1.4. 研究動機....................................................................................................12 1.5. 實驗大綱....................................................................................................13 第二章 材料與方法 2.1. 實驗材料 2.1.1. 載體....................................................................................................14 2.1.2. 大腸桿菌株....................................................................................................14 2.1.3. 大腸桿菌Kl之neuB基因來源....................................................................................................15 2.1.4. 細菌培養基....................................................................................................15 2.1.5. 去離子超純水....................................................................................................15 2.2. 實驗藥品 2.2.1. 實驗用酵素....................................................................................................16 2.2.2. 實驗用藥品....................................................................................................16 2.3. 試驗分析儀器....................................................................................................16 2.4. 分子生物學實驗 2.4.1. 染色體核酸之純化....................................................................................................18 2.4.2. 快速篩選質體....................................................................................................19 2.4.3. 小量抽取質體....................................................................................................20 2.4.4. 核酸濃度定量....................................................................................................21 2.4.5. DNA 洋菜膠體電泳法....................................................................................................21 2.4.6. DNA 片段之分離純化....................................................................................................22 2.4.7. 寡核酸引子之設計....................................................................................................23 2.4.8. 聚合?鏈鎖反應....................................................................................................24 2.4.9. 製作勝任細胞....................................................................................................26 2.4.10. 質體之切除....................................................................................................26 2.4.11. 質體與插入段之粘接....................................................................................................27 2.4.11.1. 質體pUC19 與插入段之粘接....................................................................................................28 2.4.11.2. 質體pQE30 與插入段之粘接....................................................................................................28 2.4.12. 將質體轉形至大腸桿菌 JM109..................................................................................................................28 2.4.13. 將質體轉形至大腸桿菌 Ml5....................................................................................................................29 2.4.14. 唾液酸合成?基因表現之誘導....................................................................................................30 2.4.15. 蛋白質粗抽取液之取得....................................................................................................30 2.5 蛋白質化學學實驗 2.5.1. 唾液酸合成?之純化....................................................................................................32 2.5.2 蛋白質定量法....................................................................................................32 2.5.2.1. Bradford分析法....................................................................................................32 2.5.2.2. BCA 分析法....................................................................................................33 2.5.2.3. 以 Edelhoch Method 定量已知胺基酸序列蛋白質濃度.......................................................................33 2.5.3. 聚丙烯醯胺膠體電泳.........................................................................................34 2.5.3.1. Reducing Tris-Glycine SDS-PAGE........................................................................................................34 2.5.3.2. Non-reducing Tris-Glycine SDS- PAGE.....................................................................................................35 2.5.3.3. Native Tris-GlycinePAGE................................................................................................................................35 2.5.3.4. 聚丙烯醯胺膠片的製作....................................................................................................36 2.5.3.5. Tris-Tricine SDS-PAGE....................................................................................................37 2.5.4. Antl-His6 西方墨點分析法....................................................................................................38 2.5.5. Ponceau S 蛋白質呈色....................................................................................................40 2.5.6. N端胺基酸序列分析....................................................................................................41 2.5.7. 蛋白質質譜分析....................................................................................................42 2.5.8. 蛋白質同源性與排比分析....................................................................................................42 2.5.9. N 端組氨酸序列移除....................................................................................................43 2.6. 唾液酸合成?活性分析 2.6.1. Periodate-Thiobarbituric Acid Assay...................................................................................................................44 2.6.2. 薄膜層析法....................................................................................................47 2.6.3. 核磁共振氫譜....................................................................................................48 2.7. 唾液酸合成?特性分析 2.7.1. 酵素受質專一性之測定....................................................................................................49 2.7.2. 酸鹼值對酵素之影響....................................................................................................49 2.7.3. 溫度對酵素之影響....................................................................................................49 第三章 實驗結果 3.1. 以大腸桿菌誘導表現唾液酸合成? 3.1.1. 寡核酸引子之設計....................................................................................................50 3.1.2. 聚合?連鎖反應....................................................................................................50 3.1.3. 插入段與質體 pUC19 之黏接....................................................................................................51 3.1.4. 插入段與質體 pQE30 之黏接....................................................................................................52 3.1.5. 誘導基因產物之表現....................................................................................................53 3.1.6. 唾液酸合成?之純化....................................................................................................53 3.2. 唾液酸合成?之物性分析 3.2.1. 唾液酸合成?之 SDS 膠體電泳分析....................................................................................................53 3.2.2. 不同分子量蛋白質單體之分析....................................................................................................54 3.2.2.1. 西方墨點分析法....................................................................................................54 3.2.2.2. N 端胺基酸定序....................................................................................................54 3.2.2.3. 液相管柱層析-質譜儀分析....................................................................................................55 3.2.3. 以高效能管枉層析法分離兩種不同分子量唾液酸合成?單體.............................................................55 3.2.4. 以膠體過濾法觀察唾液酸合成?雙體....................................................................................................57 3.2.5. 野生型唾液酸合成?之取得....................................................................................................58 3.3. 生物資訊-不同來源之唾液酸合成?比較 3.3.1. 同源性比較蛋白質搜尋與排比....................................................................................................59 3.3.2. 相關唾液酸合成蛋白質之演化樹分析....................................................................................................60 3.3.3. 唾液酸合成? Hydropathy 分析....................................................................................................61 3.3.4. 唾液酸合成?之多重胺基酸序列比較....................................................................................................61 3.4. 唾液酸合成?酵素反應分析 3.4.1. Periodate-Thiobarbituric Acid Assay....................................................................................................62 3.4.2. 核磁共振氫譜....................................................................................................63 3.4.3. 薄膜層析法....................................................................................................63 3.5. 唾液酸合成?特性分析 3.5.1. 酵素活性測定....................................................................................................64 3.5.2. 酵素受質專一性之測定....................................................................................................65 3.5.3. 酸鹼值對酵素之影響....................................................................................................67 3.5.4. 溫度對酵素之影響....................................................................................................67 第四章 實驗討論....................................................................................................68 第五章 未來展望....................................................................................................72 第六章 圖表與說明....................................................................................................74 第七章 參考文獻....................................................................................................108 附錄114 | |
dc.language.iso | zh-TW | |
dc.title | 選殖與大量表現大腸桿菌K1之N-乙醯基神經胺糖酸合成?及其酵素特性分析 | zh_TW |
dc.title | Cloning, Overexpression, and Characterization of N-Acetyl-D-neuraminic Acid Synthetase from Escherichia coli K1 | en |
dc.date.schoolyear | 88-2 | |
dc.description.degree | 碩士 | |
dc.relation.page | 133 | |
dc.rights.note | 未授權 | |
dc.contributor.author-dept | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
文件中的檔案:
沒有與此文件相關的檔案。
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。