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研究生:劉芫攸
研究生(外文):Yuan-Yo Liu
論文名稱:構築大豆之β-conglycininβ改造蛋白基因並在大腸菌進行表現分析
論文名稱(外文):Construction of the modified soybean β-conglycinin β genes and purification of the overexpressed recombinant proteins in Escherichia coli
指導教授:簡麗鳳簡麗鳳引用關係
指導教授(外文):Lee Feng Chien
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:95
中文關鍵詞:β-conglycinin活性胜肽VVYP蛋胺酸
外文關鍵詞:β-conglycinin活性胜肽VVYPmethionine
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大豆蛋白組成中 β-conglycinin 佔總蛋白量的 28%,共由三種次單元體 (α、α''、β) 組成三聚體結構。本研究為增加大豆功能性,並提高其附加價值,透過基因改造的方式,將降血脂胜肽 VVYP 嵌入β-conglycinin β 基因內,並利用大腸菌進行表現。先利用電腦模擬基因改造對於蛋白結構穩定性的影響,選擇 long loop 上的三個位置進行改造,藉由 crossover PCR 及引子黏合方式共得到 15 種不同的 β-conglycinin β 改造基因,分別帶有 1 到 7 套不等的 VVYP 胜肽序列,並經實驗證實這 15 種基因都能在大腸菌中表現。為改善蛋白可溶率而改以低溫條件進行表現,結果發現可溶率皆有所提升。若將改造區域之間的可溶率進行比較,以第三改造位為最佳,第五改造位次之,最後則為第七改造位。接著於第五及第七改造位置同時進行改造,得到含 8 至 11 套 VVYP 胜肽之構築,同樣在大腸菌中進行表現,發現這些高套數的構築並無可溶性蛋白產生。由於不可溶性蛋白的表現量較高,故以此型式純化這 19 種含 VVYP 胜肽之β-conglycinin β 改造蛋白,並以 trypsin 及 carboxypeptidase B 將純化蛋白水解,之後將蛋白水解液以 HPLC 進行分析,發現所釋放出之 VVYP 胜肽量與基因中所含之套數成一正比關係,而 50 μg 的 7Sbnsp-v5774 蛋白可釋放出 6972 pmol 的 VVYP 胜肽,為改造蛋白系列中 VVYP 釋放量最高者。同時也利用 LC-MS-MS和水解胺基酸組成分析來觀察胺基酸的消長並證實釋出的胜肽的序列為 VVYP。

本研究另一目的為提高β-conglycinin β 蛋白中 methionine 與 glycine 的含量,提高其作為水產飼料之潛力。因此設計了 MGKMGR 及 GMKGMR 胜肽序列,並利用改造 VVYP 之方式進行改造。共在三個改造位上得到 7 種含 MGKMGR 及 3 種含 GMKGMR 胜肽之構築,再利用大腸菌表現這 10 種改造蛋白。分析之後只有 7Sbnsp-M36、7Sbnsp-M56、7Sbnsp-M76 及 7Sbnsp-G710 有較佳的表現,之後利用 trypsin 水解並以 HPLC 分析和進行水解胺基酸組成分析,含 6 套 MGKMGR 胜肽的蛋白,能釋放出約4800 ~ 5208 pmole 的MGK 胜肽,而含 10 套 GMKGMR胜肽的蛋白,則釋放 5893 pmole 的 GMK 胜肽。但由於蛋白不穩而有降解的現象,造成這兩類胜肽的釋放率僅達預估值的 66.1% ~ 81.4%。
β-conglycinin is the second most abundant of storage proteins in soybean, which account for about 28% of the total proteins. It has a trimeric structure and composed of α, α'' and β subunits. The aim of this study is to enhance the physicochemical values of soybean seed proteins by genetic modification of β-conglycinin β with multiple copies of hypocholesterol peptide sequence (VVYP). Three sites in the variable regions (III, V and VII) of β-conglycinin β subunit suitable for genetic manipulation were predicted by computer simulation and used for construction of modified proteins. Fifteen mutants with one to seven tandem repeat of VVYP codons in one of the three variable regions were constructed either by crossover PCR or annealing of the designed primers. Four mutants have insertion in both site V and site VII and contain eight to eleven copies of VVYP codons were also constructed. All of these mutants were then transformed into E. coli and most of the recombinant proteins were expressed as inclusion bodies when the transformed bacteria were grown at 37°C. Lowered the culture temperature could enhance the solubility of recombinant proteins; however, the expression levels of recombinant proteins were significantly reduced. A total of nineteen modified β-conglycinin β proteins were then expressed in E. coli and purified from the inclusion bodies. After digesting with trypsin and carboxypeptidase B, the VVYP peptide released from the purified recombinant proteins were analyzed with HPLC. Results showed that the released VVYP peptides are in proportion to the copy numbers of VVYP sequence in the recombinant proteins. The VVYP peptides released from 7Sbnsp-v5774 protein had the highest yield among these mutants with approximately 6972 pmol peptides from 50 μg of the purified proteins. Moreover, the sequence of released peptide was confirmed to be VVYP by LC-MS-MS analysis. Compared to unmodified protein, the percentage of V, Y, P, K amino acids composition in the recombinant protein was found to increase after determination by amino acid hydrolysis.

It has been of continuing interest in identifying and developing protein source as alternatives to fish meal for use within aquafeeds. The second aim of this research was to enhance the nutritional qualities of soybean by incorporation more methionine and glycine codons. The same strategy was applied to manipulate and modified β-conglycinin β gene with MGKMGR and GMKGMR cassettes. Seven mutants containing different copies of MGKMGR cassettes and three mutants containing ten copies of GMKGMK cassettes in the β-conglycinin β gene were constructed and expressed in E. coli. SDS-PAGE analysis revealed that only recombinant proteins expressed from 7Sbnsp-M36, 7Sbnsp-M56, 7Sbnsp-M76 and 7Sbnsp-G710 mutants were detectable. These recombinant proteins were then purified from inclusion bodies and digested with trypsin for HPLC analysis. The mutant β-conglycinin β protein with six copies of MGKMGR cassettes was found to release 4800 ~ 5208 pmole peptides and 5893 pmole from that with ten copies of GMKGMR cassettes. The ratios of these two peptides released from the recombinant proteins were lower than the theoretical values and this may be explained by degradation of recombinant proteins due to protein instability.
中文摘要 IV
英文摘要 V
縮寫字對照表 VII
前言 1

材料方法
I. 材料
一、菌種及質體 10
二、藥品 10
三、純化管柱 10
四、酵素11
五、培養基及緩衝溶液11
六、引子 11

II. 實驗方法
一、大腸菌之培養 12
二、小量質體DNA製備 (Small scale preparation of plasmid) 12
三、限制酶切割反應 (Restriction enzyme digestion) 12
四、DNA 片段回收及純化 (DNA recovery) 12
五、DNA 黏合反應 (Ligation) 13
六、製備熱休克法之勝任細胞 13
七、轉形作用-熱休克法 (Transformation-heat shocked) 14
八、聚合酶鏈鎖反應 (Polymerase chain reaction;PCR) 14
九、Crossover PCR 14
十、利用核酸自動定序儀定序之質體抽取 15
十一、重組質體篩選法 15
十二、菌株甘油保存法 15
十三、蛋白質凝膠電泳分析 (SDS-PAGE) 16
十四、孔雀藍染色法 16
十五、非專一性抗體吸附 16
十六、西方墨點法 (Western blot) 16
十七、純化大腸菌生產之重組蛋白 17
十八、Ni2+-NTA 親和性管柱純化 17
十九、分析改造蛋白 β-conglycinin β 次單元體目標胜肽釋放量 18
二十、改造蛋白胺基酸組成分析 19
二十一、以 LC-MS-MS 鑑定改造蛋白所釋出的 VVYP 胜肽 19

結果

一、表現 β-conglycinin β 蛋白
1.表現含訊息序列與不含訊息序列之β-conglycinin β 蛋白 20
2.表現含訊息序列與不含訊息序列之 β-conglycinin β 改造蛋白 20

二、表現含 VVYP 序列之 β-conglycinin β 蛋白
1.電腦模擬 β-conglycinin β 重組蛋白的三級結構 21
2.改造 β-conglycinin β 基因 21
3.在大腸菌中表現含有 VVYP 胜肽之 β-conglycinin β 蛋白 24
4.純化 β-conglycinin β 及 β-conglycinin β 改造蛋白 24

三、分析 VVYP 胜肽釋放率
1.利用蛋白酶模擬消化道酵素切割 25
2.利用 HPLC 分析經酵素水解後釋出之 VVYP 胜肽 26
3.利用 LC-MS-MS 鑑定 VVYP 胜肽 27
4.利用水解分析改造蛋白 7Sbnsp 與 7Sbnsp-v5774 胺基酸組成變化27

四、表現含較高量 methionine 和 glycine 的 β-conglycinin β 改造蛋白
1.提升 β-conglycinin β 蛋白中 methionine 及 glycine 的含量 28
2.表現含有 MGKMGR 及 GMKGMR 胜肽之 β-conglycinin β 蛋白 28
3.純化各種 7Sbsp-MGKMGR 及 GMKGMR 改造蛋白4.純化定量改造蛋白及西方墨點法偵測 30
5.利用 HPLC 分析 MGK 及 GMK 胜肽的釋放率 30
6.水解分析改造蛋白 7Sbnsp-M76 及 7Sbnsp-G710 的胺基酸組成變化 32

討論
一、訊息序列對於 β-conglycinin β 蛋白在大腸菌中表現之影響 33
二、在不同培養條件下表現β-conglycinin β 蛋白 33
三、改造 β-conglycinin β 基因 34
四、在大腸菌中表現 7Sbnsp-V 蛋白 34
五、純化 7Sbnsp 蛋白與 7Sbnsp-V 改造蛋白 35
六、7Sbnsp-V 改造蛋白中 VVYP 胜肽之含量變化 35
七、7Sbnsp 與 7Sbnsp-v5774 之胺基酸組成變化 36
八、LC-MS-MS 分析 7Sbnsp-VVYP 分離之 VVYP 圖譜 37
九、利用大腸菌表現及純化 7Sbnsp-M 及 7Sbnsp-G 系列改造蛋白 37
十、7Sbnsp-M 與 7Sbnsp-G 改造蛋白中嵌入胜肽之含量變化 38
十一、7Sbnsp-M76 與 7Sbnsp-G710 之胺基酸組成變化 38

參考文獻 40
圖表 45
附錄 90
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