麩胺酸異位酶S次單元(glutamate mutase S component, MutS)是麩胺酸異位酶中的鈷胺素結合區(cobalamin-binding domain)，共含131個胺基酸。 本論文嘗試建立以化學酵素法合成此蛋白。首先，將MutS分為兩個片段分別合成： 一為從N端算起至第25個胺基酸(peptide 1)，以Fmoc固相胜肽合成法生產，於第25個胺基酸之C末端(C-terminal)處理為硫酯(thioester)； 另一部分為第26胺基酸開始至131殘基(peptide 2)。 以傳統的基因重組法將第26個胺基酸絲胺酸(serine)更改為半胱胺酸(cysteine)，同時在第26胺基酸前插入一段具有專一性的菸草蝕刻病毒蛋白酵素(tobacco etch virus NIa protease, TEV protease) 的辨識位置(E-X-L-Y-X-N↓)，轉殖入大腸桿菌中大量表現，並以TEV蛋白酵素作為切割基因重組蛋白質(His-TEV-MutS26C)的工具。 經由菸草蝕刻病毒蛋白酵素水解處理過後的蛋白質則可因此生產出N末端為半胱胺酸(cysteine)的蛋白質片段，以作為接合(ligation)的物件之一。 將上述兩個片段以自然化學接合法(native chemical ligation)將peptide 1之C端的thioester與peptide 2之 N端的半胱胺酸在中性條件下發生化學選擇之硫酯轉移反應(chemoselective transthioesterification)，在接合的位置形成新的醯胺鍵(amide bond)成一完整的蛋白質。由於化學法合成胜肽的優點，是可以引入非天然型或D型的胺基酸於產物中，以彌補一般固相胜肽合成法合成胜肽的長度有所限制的缺點。 本論文嘗試利用自然化學接合法合成分子量為14 kd的MutS次單元。

Glutamate mutase S component (MutS) is the smallest known protein subunit that carries cobalamin-binding domain with molecular masses of 14,748 Da. The chemo-enzymatic synthesis of this small monomeric protein by the native chemical ligation method is the long-term goal of in this study. Two unprotected peptide segment, 1 and 2, were synthesized separately. The segment 1 (residue 1-25) was readily prepared in good yield by solid-phase peptide synthesis. A thio-ester group was added to the carboxyl end of the segment 1. The segment 2 (residue 26-131) was produced by the over-expression of an engineered and truncated mutS gene. After treatment with TEV protease to remove the hexa-His purification tag, the segment 2 peptide carries a cysteine group at its N-terminal end. The ligation of above two fragments a rapid intramolecular S→N acyl shift through will be subsequently carried out in our group. This result indicates that incorporation of an artificial amino acid residue into the conserved cobalamin-binding motif is feasible by using this synthetic strategy.

目錄

中文摘要......................................................................................................................... i
英文摘要........................................................................................................................ ii
目錄............................................................................................................................... iii
圖目錄............................................................................................................................ v
表目錄........................................................................................................................... vi

第一章 序論.................................................................................................................. 1
   1-1 前言................................................................................................................... 1
   1-2 研究動機 ......................................................................................................... 4
   1-3 實驗設計........................................................................................................... 5

第二章 實驗部份......................................................................................................... 8
   2-1  儀器與藥品..................................................................................................... 8
   2-2  MutS表現與純化......................................................................................... 11
   2-2-1菌種培養...................................................................................................... 11
   2-2-2 破菌............................................................................................................. 11
   2-2-3 純化............................................................................................................. 12
   2-2-4 酵素水解MutS蛋白質.............................................................................. 13
   2-2-5 電漬法......................................................................................................... 14
   2-3   以固相合成法合成C-terminal thioester peptide ..................................... 15
   2-3-1 以PyBOP法接第一個胺基酸至4-sulfamylbutyryl AM resin................... 15
   2-3-2  Fmoc 胜肽固相合成法............................................................................ 16
   2-3-3  樹脂活化與移除....................................................................................... 17
   2-3-4  HPLC純化................................................................................................ 18
   2-3-5  送測質譜....................................................................................................18
   2-3-6  化學接合法............................................................................................... 19

第三章 結果與討論.................................................................................................... 20

參考文獻...................................................................................................................... 35


圖目錄

圖一、輔酶B12結構式..................................................................................................... 1
圖二、麩胺酸異構酶進行重新排列的反應作用............................................................. 2
圖三、化學合成法流程圖................................................................................................. 4
圖四、實驗設計流程圖..................................................................................................... 5
圖五、Ni2+ NTA gel 與組胺酸鍵結示意圖................................................................... 12
圖六、合成C-terminal thioester peptide之流程圖......................................................... 15
圖七、化學接合法........................................................................................................... 19
圖八、pTEV-mutS26C蛋白質表現電泳圖(Tricine SDS-PAGE)................................... 23
圖九、以Ni2+ NTA gel 純化MutS蛋白質之Tricine SDS-PAGE圖..............................24
圖十、TEV 蛋白酵素水解MutS之Tricine SDS-PAGE................................................ 25
圖十一、電漬法轉移蛋白質至PVDF膜........................................................................ 26
圖十二、protein N-terminal sequence之標準圖譜......................................................... 27
圖十三、protein N-terminal sequence residue 1.............................................................. 28
圖十四、protein N-terminal sequence residue 2.............................................................. 29
圖十五、protein N-terminal sequence residue 3.............................................................. 30
圖十六、protein N-terminal sequence residue 4.............................................................. 31
圖十七、protein N-terminal sequence residue 5.............................................................. 32
圖十八、HPLC純化α-thioester peptide........................................................................ 33
圖十九、α-thioester peptide質譜圖............................................................................... 34


表目錄

表一、生產蛋白質常用的方法......................................................................................... 3

參考文獻：
1. Holloway, D. E.; Marsh, E. N. G., Adenosylcobalamin-dependent Glutamate Mutase from Clostridium tetanomorphum. J. Biol. Chem 1994, 269, 20425-20430.
2. Holloway, D. E.; Harding, S. E.; MARSH, E. N. G., Adenosylcobalamin-dependent glutamate mutase: properties of a fusion protein in which the cobalamin-binding subunit is linked to the catalytic subunit. Biochem. J. 1996, 320, 825-830.
3. STRYER, STRYER'S 生物化學, 中文版. 86年初版一刷; p p643-644.
4. Marsh, E. N., Review Article Coenzyme-B(12)-Dependent Glutamate Mutase. Bioorg Chem. 2000, 28, (3), 176-189.
5. Hao-Ping; Lung, F.-D.; Yeh, C.-C.; Chen, H.-L.; Wu, S.-H., The role of the conserved histidine-aspartate pair in the "base-off" binding of cobalamins. Biooranic & Medicinal Chemistry 2004, 12, 577-582.
6. Kimmerlin, T.; Seebach, D., 100 years of peptide synthesis: ligation methods for peptide and protein synthesis with applications to beta-peptide assemblies. J. Peptide Res. 2005, 65, 229-260.
7. Fishcher, E.; Fourneau, E., Uebereinige Derivate des Glykokolls. Ber. Dtsch. Chem. Ges. 1901, 34, 2868-2879.
8. Merrifield, R. B., Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J.Am. Chem. Soc. 1963, 85, 2149-2154.
9. Schnölzer, M.; Kent, S. B. H., Constructing proteins by dovetailing unprotected synthetic peptides: backbone-engineered HIV protease. Science 1992, 256, 221-225.
10. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H., Synthesis of proteins by native chemical ligation. Science 1994, 266, 776-779.
11. du Vigneaud, V.; Ressler, B.; Swan, J. M.; Roberts, J. M.; Katsoyannis, C. W., The synthesis of oxytocin. J. Am. Chem. Soc. 1954, 76, 3115-3121.
12. Tolbert, T. J.; Wong, C.-H., New methods for proteomic research: preparation of proteins with N-terminal cysteines for labeling and conjugation. Angew. Chem. Int. Ed. 2002, 41, 2171-2174.
13. Tolbert, T. J.; Wong, C.-H., Intein-Mediated Synthesis of Proteins Containing Carbohydrates and Other Molecular Probes. J. Am. Chem. Soc. 2000, 122, (23), 5421-5428.
14. Muir, T. W.; Dawson, P. E.; Kent, S. B. H., Protein synthesis by chemical ligation of unprotected peptides in aqueous solution. Methods Enzymol. 1997, 289, 266-298.
15. Evans, T. C.; Benner, J. J.; Xu, M.-Q., Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci. 1998, 7, 2256-2264.
16. Marcaurelle, L. A.; Mizoue, L. S.; Wilken, J.; Oldham, L.; Kent, S. B. H.; Handel, T. M.; Bertozzi, C. R., Chemical synthesis of lymphotactin: a glycosylated chemokine with a C-terminal mucin-like domain. Chem. Eur. J. 2001, 7, 1129-1132.
17. Dougherty, W. G. S.; Cary, M.; Parks, T. D., Molecular genetic analysis of a plant virus polyprotein cleavage site: a model. Virology 1989, 171, (2), 356-364.
18. Carrington, J. C.; Dougherty., W. G., A viral cleavage site cassette: identification of amino acid sequences required for tobacco etch virus polyprotein processing. Proc. Natl. Acad. Sci. USA 1988, 85, 3391-3395.
19. Parks, T. D.; Smith, H. A.; Dougherty, W. G., Cleavage profiles of tobacco etch virus (TEV)-derived substrates mediated by precursor and processed forms of the TEV NIa proteinase. J. Gen. Virol. 1992, 73, 149-155.
20. Dougherty, W.; Semler, B., Expression of Virus-Encoded Proteinases: Functional and Structural Similarities with Cellular Enzymes. Microbiol. Rev. 1993, 57, 781-822.
21. Dougherty, W. G.; Carrington, J. C.; Cary, S. M.; Parks, T. D., Biochemical and mutational analysis of a plant virus polyprotein cleavage site. EMBO J. 1988, 7, 1281-1287.