臺灣博碩士論文加值系統

澱粉分支酶（SBE, EC 2.4.1.18）在支鏈澱粉（amylopectin）的合成上扮演重要的角色，先前已經在綠豆（Vigna radiate, cv. Tainan no. 5）中選殖出其全長之SBEⅠ cDNA（取名為VrsbeⅠ）。以生物資訊軟體預測其蛋白質3D立體結構以及特異性胺基酸序列，在Swiss PDB資料庫中比對只尋找到唯一的模板，E. coli glycogen synthase (GS) 其資料庫ID為1m7x；VrsbeⅠ對E. coli GS有27.4%的相似性，因此僅預測出VrSBEⅠ胺基酸序列中的34.3%之立體結構。在預測出之立體結構區域，在(α/β)8 domain上的八個保守性催化殘基中共有六個位於此區域。此外以REMUS互相比較綠豆之異構酶序列，可以找到VrSBEⅠ上的特異性序列，而此特異性序列則有潛力作為抗體單一性辨識VrSBEⅠ之結合位置。經定序確認過質體pET-30 EK/LIC-VrsbeI正確性後，質體轉形於表達宿主BL21（DE3）pLysS中，於LB培養液進行培養，並由IPTG誘導，誘導出目標蛋白質rVrSBEⅠ預估大小為89 kDa，而誘導時另外添加1%葡萄糖則可以減少宿主之本底表達（basal expression）。粗萃蛋白質進行amylose branching assay分析酵素活性，可於波長660位置見到吸光值之下降，顯示重組蛋白質具有SBE活性。以親和性層析管柱HisTrapTM將目標蛋白質純化，獲得之純化蛋白質其比活性為314.63 U/mg，純化倍率為114倍。

Starch branching enzyme（SBE, EC 2.4.1.18）is one of the enzymes vital for amylopectin synthesis. The sequence of the previously obtained full-length cDNA of mungbean (Vigna radiate, cv. Tainan no. 5) SBEⅠ (named VrsbeⅠ) was confirmed and cloned. Its 3-D structures and functional features were predicted in silico with the only template, E. coli glycogen synthase (GS), 1m7x, by Swiss Model and REMUS. There is 27.4% amino acid sequence of VrSBEⅠ homologous to E. coli GS, in which 34.3% 3D structure of VrSBEⅠ was predicted. Six of the 8 conserved catalytic residues within the (α/β)8 domain of the α-amylase family in VrSBEⅠ were located in the structure. When using REMUS to compare amino acid sequences between two VrSBE isoforms, there were found to be potential epitope regions which are able to be bound specifically by antibodies. After confirming codon correctness by sequencing, VrsbeⅠ was cloned into pET-30 EK/LIC expression vector. The pET-30 EK/LIC-VrsbeI was expressed in BL21（DE3）pLysS cells in standard LB broth and the protein was induced by IPTG. The recombinant enzyme, rVrSBEⅠ, had His-tag and S-tag at the N terminal with an estimated molecular mass of 89 kDa. When Supplied with an extra 1% Glucose as carbon source during induction, it was able to decrease basal protein expression in the E. coli host. The crude cell extract possessing branching enzyme catalytic activities that decrease the A660 absorbance of amylose-iodine complex indicated that rVrSBEⅠ protein would be expressed as an active form. The crude extract was purified by HisTrapTM affinity chromatography. The activity of the purified rVrSBEI was also assayed by amylose branching assay. The decrease of the absorbance at A660 exhibited the specific activity of rVrSBEI was 314.6 U/mg and the purity has enriched 114-fold.

總目錄
總目錄………………………………………………………………Ⅳ
圖目錄………………………………………………………………Ⅵ
表目錄………………………………………………………………Ⅹ

中文摘要...................................................1
英文摘要...................................................2
第一章 序論.........................................................3
第一節 澱粉生合成作用......................................3
1.1 澱粉的組成及結構.......................................3
1.2 澱粉的生合成與參與酵素.................................4
第二節 澱粉分支酶之分子生物學研究..........................6
2.1 澱粉分支酶分別為famility A與B二大類....................7
第三節 綠豆簡介...........................................10
第四節 sbeⅠ、sbeⅡ特性差異...............................13
第五節 研究起源與目的.....................................14
第二章 材料與方法.........................................15
第一節 材料........................................................15
1.1 樣品..................................................15
1.2 藥品..................................................16
1.3 儀器設備..............................................17
第二節 實驗流程設計.......................................19
第三節 聚合酶連鎖反應.....................................20
第四節 核酸電泳分析法.....................................26
第五節　DNA片段之純化方法.................................27
第六節 EK/LIC pET-30 Vector之轉殖.........................29
第七節 Plasmid DNA之抽取..................................35
第八節 DNA序列定序........................................36
第九節 重組蛋白質誘導流程.................................37
第十節 表現蛋白質的分析...................................38
10.1 表現蛋白質的萃取與純化...............................38
A. 萃取蛋白質......................................38
B. 純化蛋白質......................................41
10.2 表現蛋白質的定量.....................................44
10.3 表現蛋白質的電泳分析.................................44
10.4 蛋白質染色法.........................................47
第十一節 SBE活性測試......................................48
Amylose-branching assay.............................48
第三章 結果與討論.........................................50
A. VrSBEⅠ胺基酸序列結構預測.......................50
B. VrsbeⅠ之PCR量化與片段純化......................52
C. 質體挑選與定序..................................53
D. 目標蛋白質表現與萃取............................55
E. 目標蛋白質之純化................................57
F. rVrSBEⅠ活性分析................................60
第四章 總結...............................................62
第五章 結果圖表...........................................63
第六章 附圖與表...........................................93
參考文獻.................................................100

圖目錄
圖一、澱粉的分支結構......................................93
圖二、DBE澱粉去分支酶在支鏈澱粉生合成功能被提出之作用模型.94
圖三、澱粉的生合成由光合作用產物蔗糖開始的路徑圖..........95
圖四、各物種sbe及其基因家族….............................96
圖五、VrsbeI cDNA及其演繹出之胺基酸序列對照...............98
圖六、KOD Hot Start DNA polymerase正確性及合成速率比較....21
圖七、常使用的hot start systems其專一性及靈敏度比較.......22
圖八、pET-30 EK/LIC載體結構圖.............................33
圖九、EK/LIC insert黏合示意圖.............................34
圖十、E. coli glycogen synthase之3D立體結構作為預測VrSBEⅠ的模板......................................................63
圖十一、VrSBEⅠ之unique sequences相對應於其3D立體預測結構上之位置....................................................63
圖十二、E. coli GS及VrSBEⅠ中心催化區域保留性殘基之3D結構圖........................................................64
圖十三、以REMUS比對出獨特loop之胜肽鏈於預測結構上之位置圖........................................................65
圖十四、使用不同原始VrsbeⅠcDNA模板含量以F9及R21引子進行PCR擴增之產物電泳圖..........................................66
圖十五、VrsbeⅠ以LIC引子進行插入子片段之二次PCR擴增及其一次PCR產物自膠體萃取純化出單一片段當作二次PCR模板之電泳圖....67
圖十六、PCR clean up純化由VrsbeⅠ以LIC引子所PCR擴增產物之電泳圖......................................................68
圖十七、膠體純化之VrsbeⅠ/LIC插入片段接合入pET-30 EK/LIC載體後轉形至選殖宿主Novablue獲得之positive clone..............69
圖十八、PCR clean up純化之VrsbeⅠ/LIC插入片段接合入pET-30 EK/LIC載體後轉形至選殖宿主Novablue獲得之positive clone....69
圖十九、兩種插入子純化方法所獲之Novablue positive colonies的劃線培養........................................................70
圖二十、由14個positive colonies抽取plasmid DNA進行電泳分析圖........................................................71
圖二十一、含有質體DNA大小在6~8 kbp組之positive colonies的劃線培養........................................................72
圖二十二、以限制酶切割確認帶有VrsbeⅠ postive clone之質體.73
圖二十三、四組positive clone之質體DNA以LIC primers進行PCR量化確認之電泳圖............................................74
圖二十四、VrsbeⅠ序列內部不同之引子組對pET-30 EK/LIC- VrsbeⅠ進行不同片段大小量化確認之電泳圖........................75
圖二十五、pET-30 EK/LIC-VrsbeⅠ-1定序結果與VrsbeⅠ序列進行並列比對....................................................77
圖二十六、pET-30 EK/LIC-VrsbeⅠ-2定序結果與VrsbeⅠ序列進行並列比對....................................................79
圖二十七、、pET-30 EK/LIC-VrsbeⅠ-1轉譯出之胺基酸序列與VrsbeⅠ之胺基酸序列進行並列比對................................80
圖二十八、pVrsbeⅠ-2轉形至表達宿主BL21 (DE3) pLysS之positive clone培養皿......................................81
圖二十九、隨機挑選六個positive colony之劃線次培養.........81
圖三十、挑選BL21 (DE3) pLysS的六個positive colonies以LIC引子對進行colony PCR 確認pVrsbeⅠ轉形之電泳圖.................82
圖三十一、不同培養液成份對重組蛋白質rVrSBEⅠ表現之電泳分析83
圖三十二、不同濃度IPTG誘導濃度對重組蛋白質rVrSBEⅠ表現之電泳分析......................................................85
圖三十三、以BugBuster及sonication方法萃取之粗萃重組蛋白質之電泳圖....................................................86
圖三十四、利用注射針筒方式將粗萃蛋白質樣品以HisTrapTM HP管柱純化後收集區分以Silver Staining染色之電泳圖...............87
圖三十五、以HisTrapTM HP管柱於AKTA prime層析系統進行重組蛋白質rVrSBEⅠ純化之層析圖譜..................................88
圖三十六、以HisTrapTM HP管柱於AKTA prime層析系統進行重組蛋白質rVrSBEⅠ純化所收集之區分的coomassie blue staining電泳圖.89
圖三十七、以HisTrapTM HP管柱於AKTA prime層析系統進行重組蛋白質rVrSBEⅠ純化所收集之區分的Silver Staining電泳圖.........90
圖三十八、粗萃重組蛋白質rVrSBEⅠ以amylose branching assay之活性測試..................................................91
圖三十九、純化後之重組蛋白質rVrSBEⅠ以amylose branching assay之活性測試...........................................92

表目錄
表一、VrSBE與其他物種BE的胺基酸序列保守性區域列表.........99
表二、VrSBE與其他物種BE的胺基酸序列保守性區域列表.........99
表三、E. coli GS與VrSBEⅠ保守性活性催化殘基位置列表......100
表四、本探討之VrsbeⅠ所使用的基因特異性引子列表...........65
表五、不同IPTG誘導濃度之BL21 (DE3) pLysS細胞之pellet濕重與soluble protein濃度.......................................84
表六、rVrSBEⅠ純化表......................................92

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