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研究生:蕭斐雅
研究生(外文):Hsiao Fei-ya
論文名稱:利用組合式資料庫研究蛋白酶之酵素特異性
論文名稱(外文):Combinatorial approach to investigate enzyme specificities of protease
指導教授:張文章陳水田陳水田引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:89
中文關鍵詞:組合式資料庫蛋白□
外文關鍵詞:combinatorial libraryprotease
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固相□□合成法為Merrifield於1963年所提出。 這項技術不但使□□合成變得快速且簡便, 更被應用到許多地方, 尤其,近十五年來,固相合成法被大量的應用在合成組合式資料庫上,組合式資料庫可以從數千種合成的混合物中挑出具有生物活性的化合物,對於找尋具有生物活性之物質是一個有效的方法。本論文的目的在利用組合式□□資料庫篩選蛋白□之酵素特異性。
我們利用固相□□合成法合成一個內含只有P1位置作變異的□□衍生物的資料庫,經蛋白□催化水解反應後,以高效能液相層析儀分析出水解反應最快的成分,以決定該蛋白質最適合P1位置的胺基酸殘基,之後利用"分裂-連結-混合"法,固定所找到之P1位置胺基酸殘基進一步合成改變P2位置胺酸殘基的□□資料庫,再進行同樣的蛋白□催化水解反應,找到最適合P2位置之胺基酸殘基。
在實驗中先取幾個已知其受質專一性的蛋白□:胰蛋白□、胰凝乳蛋白□、離胺酸水解□及麩胺酸水解□進行資料庫之正確性的試驗,證明資料庫的適用性無誤後,取未知受質專一性的蛋白□TM-1篩選P1及P2位置最適合之胺基酸殘基。實驗結果顯示TM-1在P1位置偏好側鏈帶有苯環的苯丙胺酸(Phe)及酪胺酸(Tyr),以這兩個胺基酸殘基分別用"分裂-連結-混合"法的結果合成P2位置作變異的資料庫,對TM-1篩選,其實驗結果為TM-1偏好在P2位置為疏水性胺基酸殘基。因此一旦成功的建立一個合適的組合式資料庫,並找到適合的分析條件後,即可快速研究出蛋白□之酵素特異性。
The solid phase peptide synthesis method was proposed by Merrfield in 1963. The technology was not only used to make peptide synthesis faster and more simply, but also applied to many other fields. Most of all, solid phase synthesis was applied to synthesize combinatorial library recently. Combinatorial library is a powerful tool to find out active compounds from a mixture of thousands compounds. It is the efficient approach for finding the biologically active molecules. The main aim of the thesis is to establish a simple combinatorial peptide library for screening the substrate specificities of proteases.
We constructed a library of tetrapeptide-ester in which all the compounds were just different in the P1 site. The protease will catalyze the hydrolysis of peptide-esters that have the suitable amino acid residue in the P1 site and release tetrapeptide from the peptide-ester library. By using high performance liquid chromatography as monitor, we could find out the peptide-ester with faster hydrolysis rate. According to the most suitable amino residue of the P1 site, the peptide-ester library in which were different in the P2 site was synthesized by the〝split-couple-mix〞method and repeat the protease-catalyzed hydrolysis reaction to screen the suitable amino acid residue of P2 site.
Before the specificity studies of unknown protease, the accuracy of the library was tested by the proteases which their specificities were well known.The trypsin, chymotrypsin, endoproteinase Lys-C and endoproteinase Glu-C were chosen for the test, and the experimental results shown that the peptide-ester library we constructed was suitable for the enzyme specificity studies. After confirming the accuracy of the library, we used the library to study the protease TM-1 which the specificity was unknown and found that its specificity in P1 site prefers phenylalanine and tyrosine, both have phenyl group at their side chain. Based on the P1 site amino acid residue, we synthesized peptide libraries which changed the amino acid residue in the P2 site by〝split-couple-mix〞method and studied the P2 site of TM-1.We found that TM-1 prefers hydrophobic amino acid residues at P2 site.
Overall, we show that the combinatorial library approach is a simple method to investigate specificity.
目錄 ………………………………………….…………………………..…..… I
圖目錄 …………………………………………………………………………Ⅲ
表目錄 …………………………………………………………………………Ⅴ
中文摘要 ………………………………………………………………………Ⅵ
英文摘要 ………………………………………………………………………Ⅶ
第一章緒論
一、 組合式資料庫 ………………………………………………..………1
二、 固相合成法 …….…………………………………………….…6
三、 蛋白之簡介 ……………….………………………………….…..11
四、 酵素特異性 ……………………………………………………...….14
第二章實驗動機及目的
一、 實驗動機 ……………………………………………………….. ….17
二、 實驗目的 ……………………………………………………..……..18
第三章實驗試劑及儀器
一、 實驗試劑 ………………………………………………...…….……19
二、 實驗儀器 ……………………………………………………….….. 21
三、 藥品溶液的配製 ……………………………..………………..……22
第四章實驗方法
一、 實驗設計 ………………………………………….……………….. 24
二、 化學合成法合成胺基酸衍生物 ………………….……………….. 26
三、 固相合成法合成資料庫 ………………………….………….. 27
四、 高效能液相層析 (HPLC) 分析方法 ………………….….………. 31
五、 蛋白催化反應條件 ……………………………….….………….32
第五章結果與討論
一、胺基酸衍生物之合成及純化 ………………………………………….34
二、P1資料庫之合成及純化 …………………………………………37
三、尋找資料庫最適合分析的紫外光吸收波長及HPLC分析條件 ….….39
四、測試資料庫的正確性 …………………………………………….43
五、TM-1與P1資料庫作用之結果 …………………………..……….56
六、"分裂-連結-混合"法合成P2資料庫 ………………………….………..60
七、TM-1與P2資料庫作用之結果 …………..………………..…….62
第六章結論 …………………………………………………………………..69
參考文獻 ……………………………………………………………….…………..71
附錄
圖目錄
圖1-1 資料庫的建構方法 …………………………………….. ………2
圖1-2 資料庫表現於絲狀菌體表面 ………………………………..….3
圖1-3 "一樹脂及一化合物" 法 ………………………………………….….4
圖1-4 以Fmoc為保護基時,固相合成之流程圖 ……………………8
圖1-5 以Boc為保護基時,固相合成之流程圖 ……………………….9
圖1-6 Kaiser test的反應機制 ………………………..………………….…..10
圖1-7 蛋白與受質結合部位圖解 ...……………………………………...14
圖4-1 實驗進行的流程圖 ……………………………………………….…25
圖4-2 Fmoc合成法切離條件選擇流程圖 ………………….……..…29
圖5-1 核磁共振氫譜之理論值 ………………………………………….…35
圖5-2 紫外光掃描 ……………………………………….…………………40
圖5-3 Suc-Ala-Ala-Ala-Gly-OR 水解前後HPLC圖 ………………………41
圖5-4 Suc-Ala-Ala-Ala-Phe-OR 水解前後HPLC圖譜 …………………….41 圖5-5 P1資料庫之HPLC分析圖譜 ………………………..……………...42
圖5-6 P1資料庫在胰蛋白水解反應緩衝液之空白實驗 ……………….44
圖5-7 P1資料庫在胰蛋白水解反應之HPLC分析圖譜 ………………..45
圖5-8 P1資料庫在胰蛋白水解反應之HPLC吸收峰面積積分曲線圖...46
圖5-9 P1資料庫在胰凝乳蛋白水解反應之HPLC分析圖譜 …………..47
圖5-10 P1資料庫在胰凝乳蛋白水解反應之HPLC吸收峰面積積
分曲線圖 …………………………………………………………….48
圖5-11 P1資料庫在離胺酸水解水解反應緩衝液之空白實驗 ……...….50
圖5-12 P1資料庫在離胺酸水解水解反應之HPLC分析圖譜 ………….51
圖5-13 P1資料庫在離胺酸水解水解反應之HPLC吸收峰面積積
分曲線圖 ………………………………………………………...…..52
圖5-14 P1資料庫在麩胺酸水解水解反應之HPLC分析圖 ……………53
圖5-15 P1資料庫在麩胺酸水解水解反應之HPLC吸收峰面積
積分曲線圖 ………………………………………………………….54
圖5-16 P1資料庫在TM-1水解反應緩衝液之空白實驗 ……………….….55
圖5-17 P1資料庫在TM-1催化水解反應之HPLC分析圖譜 …………..…56
圖5-18 P1資料庫在TM-1催化水解反應之HPLC吸收峰面積
積分曲線圖 ……………………………………………………….…57
圖5-19 F資料庫之HPLC分析圖譜 …………………………………...……58
圖5-20 Y資料庫之HPLC分析圖譜 …………………………………....…..59
圖5-21 F-A子資料庫在TM-1催化水解反應之HPLC分析圖譜 ……....…61
圖5-22 F-Y子資料庫在TM-1催化水解反應之HPLC分析圖譜 ………....62
圖5-23 F資料庫在TM-1催化水解反應之HPLC吸收峰面積
積分曲線圖 ………………………………………………………….63
圖5-24 Y-A子資料庫在TM-1催化水解反應之HPLC分析圖 ……………65
圖5-25 Y-Y子資料庫在TM-1催化水解反應之HPLC分析圖譜 ………...66
圖5-26 Y資料庫在TM-1催化水解反應之HPLC吸收峰面積積分
曲線圖 ………………………………………………………….……67
圖5-27 Y資料庫及F資料庫在TM-1催化水解反應之HPLC吸收
峰面積積分曲線圖 …………………………………………...……..68
表目錄
表1-1 利用反應催化模式來分別蛋白的種類………………………….. …12
表1-2 利用反應催化機制來分別蛋白的種類 …………...…………….…..13
表1-3本實驗中所使用之蛋白列表 ..…………………………………….…15
表5-1 胺基酸酯類衍生物之物性 …………………………………………......36
表5-2 酯類衍生物之物性 ………………………………………………...…...38
表5-3 P2資料庫的分類及命名 …………………………………………….…58
1. Bartel, D. P. & Szostak, J. W. (1993) Isolation of new ribozymes from a large pool of random sequences, Science. 261, 1411-8.
2. Joyce, G. F. (1989) Amplification, mutation and selection of catalytic RNA, Gene. 82, 83-7.
3. Breaker, R. R. & Joyce, G. F. (1994) A DNA enzyme that cleaves RNA, Chem Biol. 1, 223-9.
4. Griffin, L. C., Toole, J. J. & Leung, L. L. (1993) The discovery and characterization of a novel nucleotide-based thrombin inhibitor, Gene. 137, 25-31.
5. Scott, J. K. & Craig, L. (1994) Random peptide libraries, Curr Opin Biotechnol. 5, 40-8.
6. Blond Elguindi, S., Cwirla, S. E., Dower, W. J., Lipshutz, R. J., Sprang, S. R., Sambrook, J. F. & Gething, M. J. (1993) Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP, Cell. 75, 717-28.
7. Liang, R., Yan, L., Loebach, J., Ge, M., Uozumi, Y., Sekanina, K., Horan, N., Gildersleeve, J., Thompson, C., Smith, A., Biswas, K., Still, W. C. & Kahne, D. (1996) Parallel synthesis and screening of a solid phase carbohydrate library, Science. 274, 1520-2.
8. Tseng, K., Hedrick, J. L. & Lebrilla, C. B. (1999) Catalog-library approach for the rapid and sensitive structural elucidation of oligosaccharides, Anal Chem. 71, 3747-54.
9. Sofia, M. J. (1998) Carbohydrate-based combinatorial libraries, Mol Divers. 3, 75-94.
10. Nefzi, A., Dooley, C., Ostresh, J. M. & Houghten, R. A. (1998) Combinatorial chemistry: from peptides and peptidomimetics to small organic and heterocyclic compounds, Bioorg Med Chem Lett. 8, 2273-8.
11. Tietze, L. F. & Lieb, M. E. (1998) Domino reactions for library synthesis of small molecules in combinatorial chemistry, Curr Opin Chem Biol. 2, 363-71.
12. Carell, T., Wintner, E. A., Sutherland, A. J., Rebek, J., Jr., Dunayevskiy, Y. M. & Vouros, P. (1995) New promise in combinatorial chemistry: synthesis, characterization, and screening of small-molecule libraries in solution, Chem Biol. 2, 171-83.
13. Dankwardt, S. M., Phan, T. M. & Krstenansky, J. L. (1996) Combinatorial synthesis of small-molecule libraries using 3-amino-5-hydroxybenzoic acid, Mol Divers. 1, 113-20.
14. Atuegbu, A., Maclean, D., Nguyen, C., Gordon, E. M. & Jacobs, J. W. (1996) Combinatorial modification of natural products: preparation of unencoded and encoded libraries of Rauwolfia alkaloids, Bioorg Med Chem. 4, 1097-106.
15. Geysen, H. M., Meloen, R. H. & Barteling, S. J. (1984) Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid, Proc Natl Acad Sci U S A. 81, 3998-4002.
16. Houghten, R. A., Pinilla, C., Blondelle, S. E., Appel, J. R., Dooley, C. T. & Cuervo, J. H. (1991) Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery, Nature. 354, 84-6.
17. Furka, A. A., Sebestyen, F., Asgedom, M. & Dibo, G. Abstracts of the 14th International Congress of Biochemistry,Prague,July 10-15.
18. al-Obeidi, F., Hruby, V. J. & Sawyer, T. K. (1998) Peptide and peptidomimetic libraries. Molecular diversity and drug design, Mol Biotechnol. 9, 205-23.
19. Cwirla, S. E., Peters, E. A., Barrett, R. W. & Dower, W. J. (1990) Peptides on phage: a vast library of peptides for identifying ligands, Proc Natl Acad Sci U S A. 87, 6378-82.
20. Devlin, J. J., Panganiban, L. C. & Devlin, P. E. (1990) Random peptide libraries: a source of specific protein binding molecules, Science. 249, 404-6.
21. Scott, J. K. & Smith, G. P. (1990) Searching for peptide ligands with an epitope library, Science. 249, 386-90.
22. Lam, K. S., Salmon, S. E., Hersh, E. M., Hruby, V. J., Kazmierski, W. M. & Knapp, R. J. (1991) A new type of synthetic peptide library for identifying ligand-binding activity , Nature. 354, 82-4.
23. Jacobs, J. W. & Fodor, S. P. (1994) Combinatorial chemistry--applications of light-directed chemical synthesis, Trends Biotechnol. 12, 19-26.
24. Schultz, J. S. (1996) The combinatorial library: a multifunctional resource, Biotechnol Prog. 12, 729-43.
25. Dodd, J. H., Schwender, C. F., Conley, R. A., Moore, J. B., Jr., Ritchie, D. M., Fabian, A. C., Fenk, C. J., Bourcet, E., Gray Nunez, Y., Kirchner, T. & et al. (1993) Synthesis and biological properties of RWJ 22108, a bronchoselective calcium channel blocker, Drug Des Discov. 10, 65-75.
26. Blondelle, S. E., Perez Paya, E. & Houghten, R. A. (1996) Synthetic combinatorial libraries: novel discovery strategy for identification of antimicrobial agents, Antimicrob Agents Chemother. 40, 1067-71.
27. Blondelle, S. E. & Houghten, R. A. (1996) Novel antimicrobial compounds identified using synthetic combinatorial library technology, Trends Biotechnol. 14, 60-5.
28. Ostresh, J. M., Blondelle, S. E., Dorner, B. & Houghten, R. A. (1996) Generation and use of nonsupport-bound peptide and peptidomimetic combinatorial libraries, Methods Enzymol. 267, 220-34.
29. Gundlach, B. R., Wiesmuller, K. H., Junt, T., Kienle, S., Jung, G. & Walden, P. (1996) Determination of T cell epitopes with random peptide libraries, J Immunol Methods. 192, 149-55.
30. Zhou, S., Carraway, K. L., 3rd, Eck, M. J., Harrison, S. C., Feldman, R. A., Mohammadi, M., Schlessinger, J., Hubbard, S. R., Smith, D. P., Eng, C. & et al. (1995) Catalytic specificity of protein-tyrosine kinases is critical for selective signalling , Nature. 373, 536-9.
31. Merrifield, R. B. (1963) Solid Phase Peptide Synthesis. Ⅰ. The Synthesis of Tetrapeptide, J. Am. Chem. Soc. 85, 2149-2154.
32. Merrifield, R. B., Stewart, J. M. & Jernberg, N. (1966) Instrument for automated synthesis of peptides, Anal Chem. 38, 1905-14.
33. Curtius, T. J. (1881) J Pract Chem. 24, 239.
34. Fischer, E. (1902) Ber Deutsch Chem Ges. 35, 1095.
35. Bergamann, M. & Zervas, L. (1932) Ber Dt Chem Ges. 65, 1192.
36. Vigeneaud, V. D., Ressler, C., Swan, J. M., Roberts, C. W., Katsoyannis, P. G. & Gordon, S. (1953) J Am Chem Soc. 75, 4879.
37. Kent, S. B. (1988) Chemical synthesis of peptides and proteins, Annu Rev Biochem. 57, 957-89.
38. Satyanarayana, J., Gururaja, T. L., Naganagowda, G. A., Ramasubbu, N. & Levine, M. J. (1998) A concise methodology for the stereoselective synthesis of O-glycosylated amino acid building blocks: complete 1H NMR assignments and their application in solid-phase glycopeptide synthesis, J Pept Res. 52, 165-79.
39. DeOgny, L., Pramanik, B. C., Arndt, L. L., Jones, J. D., Rush, J., Slaughter, C. A., Radolf, J. D. & Norgard, M. V. (1994) Solid-phase synthesis of biologically active lipopeptides as analogs for spirochetal lipoproteins, Pept Res. 7, 91-7.
40. Ito, Y. & Manabe, S. (1998) Solid-phase oligosaccharide synthesis and related technologies, Curr Opin Chem Biol. 2, 701-8.
41. Bauser, M., Winter, M., Valenti, C. A., Wiesmuller, K. H. & Jung, G. (1998) Synthesis of hydantoins via N,N''-ureas derived from polymer-bound amino acids, Mol Divers. 3, 257-60.
42. Linkletter, B. A. & Bruice, T. C. (1998) Solid-phase synthesis of oligomeric deoxynucleic guanidine (DNG): a polycationic analogue of DNA, Bioorg Med Chem Lett. 8, 1285-90.
43. Trautwein, A. W., Sussmuth, R. D. & Jung, G. (1998) Hantzsch pyrrole synthesis on solid support, Bioorg Med Chem Lett. 8, 2381-4.
44. Kim, S. W., Hong, C. Y., Lee, K., Lee, E. J. & Koh, J. S. (1998) Solid phase synthesis of benzylamine-derived sulfonamide library, Bioorg Med Chem Lett. 8, 735-8.
45. Boeijen, A., Kruijtzer, J. A. & Liskamp, R. M. (1998) Combinatorial chemistry of hydantoins, Bioorg Med Chem Lett. 8, 2375-80.
46. Smith, R. A., Bobko, M. A. & Lee, W. (1998) Solid-phase synthesis of a library of piperazinediones and diazepinediones via Kaiser oxime resin, Bioorg Med Chem Lett. 8, 2369-74.
47. Chickos, J. S. (1970) The 9-Fluorenylmethoxycarbonyl Function,a New Base-Sensitive Amino- Protecting group, J. Am. Chem. Soc. 92, 5748-5750.
48. Carpino, L. A. (1957) Oxidative Reactions of Hydrazines. Ⅳ. Elimination of Nitrogen from 1,1-Disubstituted-2-arenesulfonhydrazides,J Am Chem Soc. 79, 4427-4431.
49. Carpino, L. A. & Han, G. Y. (1972) The 9-Fluorenylmethoxycarbonyl Amino-Protecting Group, J Org Chem. 37, 3404-3409.
50. Guy, C. A. & Fields, G. B. (1997) Trifluoroacetic acid cleavage and deprotection of resin-bound peptides following synthesis by Fmoc chemistry, Methods Enzymol. 289, 67-83.
51. Carpino, L. A. (1956) Oxidative Reactions of Hydrazines. Ⅱ. Isophthalimides. New Protective Groups Nitrogen, J Am Chem Soc. 79, 98-101.
52. Stewart, J. M. (1997) Cleavage methods following Boc-based solid-phase peptide synthesis, Methods Enzymol. 289, 29-44.
53. Ranganathan, S., Ranganathan, D. & Methrotra, A. K. (1975) An unanticipated, facile,regiospecific and stereospecific A → F prostanoid transformation, Tetrahedron letters. 14, 1214-1216.
54. Knorr, R., Trzeciak, A., Bannwarth, W. & Gillessen, D. (1989) New Coupling in Peptide Chemistry, Tetrahedron Letters. 30, 1927-1930.
55. Carpino, L. A. (1993) 1-Hydroxy-7-azabenzotriazole. An Efficient Peptide Coupling Additive, J Am Chem Soc. 115, 4397-4398.
56. Kaiser, E., Colescott, R. L., Bossinger, C. D. & Cook, P. I. (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides, Anal Biochem. 34, 595-8.
57. McFerran, N. V., Walker, B., McGurk, C. D. & Scott, F. C. (1991) Conductance measurements in solid phase peptide synthesis. I. Monitoring coupling and deprotection in Fmoc chemistry, Int J Pept Protein Res. 37, 382-7.
58. Atherton, E. & Sheppard, R. C. (1989) in Solid Phase Peptide Synthesis:A Practical Approach., 112-117.
59. Linderstrom-Lang, K. (1929) Z Physiol Chem. 182, 151.
60. Barrett, A. J. (1994) Classification of peptidases, Methods Enzymol. 244, 1-15.
61. Fischer, E. (1894) Ber Dt Chem Ges. 27, 2985-2991.
62. Koshland, D. E. J. (1958) Application of a theory of enzyme specificity to protein synthesis., Proc Natl Acad Sci USA. 44, 98-123.
63. Schechter, I. & Berger, A. (1967) On the size of the active site in proteases. I. Papain, Biochem Biophys Res Commun. 27, 157-62.
64. Riordan, J. F., Wacker, W. E. & Vallee, B. L. (1965) "Buried" tyrosyl residues and the activity of trypsin, Nature. 208, 1209-11.
65. Tomasek, V., Severin, E. S. & Sorm, F. (1965) Studies on the active site of trypsin. II. The role of the imidazole ring of histidine in the catalytic action of trypsin, Biochem Biophys Res Commun. 20, 545-9.
66. Russo, S. F. & Holzman, T. (1977) The activity of trypsin, J Chem Educ. 54, 60-1.
67. Blow, D. M. (1968) Chymotrypsin: tertiary structure and enzymic activity, Biochem J. 110, 2P.
68. Jekel, P. A., Weijer, W. J. & Beintema, J. J. (1983) Use of endoproteinase Lys-C from Lysobacter enzymogenes in protein sequence analysis, Anal Biochem. 134, 347-54.
69. Drapeau, G. R. (1978) The primary structure of staphylococcal protease, Can J Biochem. 56, 534-44.
70. Huang, K. F., Hung, C. C. & Chiou, S. H. (1993) Characterization of three fibrinogenolytic proteases isolated from the venom of Taiwan habu (Trimeresurus mucrosquamatus), Biochem Mol Biol Int. 31, 1041-50.
71. Huang, K. F., Hung, C. C., Pan, F. M., Chow, L. P., Tsugita, A. & Chiou, S. H. (1995) Characterization of multiple metalloproteinases with fibrinogenolytic activity from the venom of Taiwan habu (Trimeresurus mucrosquamatus): protein microsequencing coupled with cDNA sequence analysis, Biochem Biophys Res Commun. 216, 223-33.
72. Ownby, C. L., Bjarnason, J. & Tu, A. T. (1978) Hemorrhagic toxins from rattlesnake (Crotalus atrox) venom. Pathogenesis of hemorrhage induced by three purified toxins, Am J Pathol. 93, 201-18.4431.
73. Sorensen, S. B., Sorensen, T. L. & Breddam, K. (1991) Fragmentation of proteins by S. aureus strain V8 protease. Ammonium bicarbonate strongly inhibits the enzyme but does not improve the selectivity for glutamic acid, FEBS Lett. 294, 195-7.
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