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偽產鹼假單胞菌F-111脂解酶之分子伴護基因選殖及特性研究 = Gene...

國立高雄大學生命科學系碩士班

偽產鹼假單胞菌F-111脂解酶之分子伴護基因選殖及特性研究 = Gene cloning and characterization of lipase chaperone from Pseudomonas pseudoalcaligenes F-111

紀錄類型:	書目-語言資料,印刷品 : 單行本
並列題名:	Gene cloning and characterization of lipase chaperone from Pseudomonas pseudoalcaligenes F-111
作者:	李韋佑,
其他團體作者:	國立高雄大學
出版地:	[高雄市]
出版者:	撰者;
出版年:	2015[民104]
面頁冊數:	64面圖，表 : 30公分;
標題:	分子伴護
標題:	Chaperone
電子資源:	http://handle.ncl.edu.tw/11296/ndltd/97393148316717218229
附註:	104年10月31日公開
附註:	參考書目：面44-50
摘要註:	大腸桿菌(Escherichia coli)在現代生物工程中經常被用作基因選殖以及生產目標蛋白質的宿主。然而,由於格蘭氏陰性菌細胞膜的內、外膜雙層結構,致使目標蛋白質往往會堆積於雙層膜之間的周質(periplasm)空間,形成不具生物活性的包涵體(inclusion body)。本篇研究期望能以Pseudomonas pseudoalcaligenes F-111菌株的脂解酶分子伴護,來研究其對於F-111脂解酶能否分泌至細胞外所扮演的角色。以PCR技術將F-111的脂解酶以及脂解酶分子伴護基因片段選殖到載體pGEM-3zf(+),電腦軟體預測顯示所選殖的脂解酶基因具備有胞外分泌所需的信號肽；並以同源模擬的方式預測所選殖的脂解酶分子伴護基因的蛋白質結構。將目標基因以E. coli表現系統進行蛋白質表現之後發現,具備完整脂解酶以及脂解酶分子伴護基因片段的重組質體能表現出脂解酶活性;然而,若將此重組質體上的脂解酶分子伴護基因剔除,儘管SDS-PAGE證實仍有脂解酶分子的表現,但是在進行活性分析時卻無法如原先的實驗結果觀察測得脂解酶活性。綜合實驗結果,研究所選殖出的脂解酶分子伴護基因,對於脂解酶的活性確實有著決定性的影響。期望此項發現能於未來建構帶有此段分子伴護基因之DNA表現載體,使大腸桿菌表現系統原本目標蛋白容易形成包涵體的問題,能在脂解酶分子伴護的協助下,便能順利送出細胞外,以增進蛋白質純化的方便性。 Escherichia coli is a commonly used gene transformation and protein production host in modern bioengineering. However, its Gram-negative double layer membrane structure also causes target protein aggregation in its periplasm space, leading to a result of inactive inclusion body formation. The aim of this study is to utilize a bacterial strain Pseudomonas pseudoalcaligenes F-111 to study its lipase chaperone's characteristics of assisting lipase secretion.By utilizing PCR, both the lipase and lipase chaperone genes from F-111 were cloned and ligated with vector pGEM-3zf(+). The computer bioinformatics prediction showed the lipase polypeptide contains a signal peptide, a necessary for secretion. Meanwhile, the structure of lipase chaperone was predicted by homology modeling. Protein analysis showed the E. coli strain transformed with complete lipase and lipase chaperone genes express lipase activities. However, if the lipase chaperone gene is knocked out, despite SDS-PAGE still shows lipase expression, it did not express its activity as the E. coli strain transformed with complete lipase and lipase chaperone genes. In conclusion, the lipase chaperone gene cloned in this study plays a crucial role in lipase activity. The study expects the discovery would lead to constructing an expression vector with the lipase chaperone gene attached for it assisting target protein secreting, to improve the convenience of protein purification in future.

偽產鹼假單胞菌F-111脂解酶之分子伴護基因選殖及特性研究 = Gene cloning and characterization of lipase chaperone from Pseudomonas pseudoalcaligenes F-111
李, 韋佑

偽產鹼假單胞菌F-111脂解酶之分子伴護基因選殖及特性研究 = Gene cloning and characterization of lipase chaperone from Pseudomonas pseudoalcaligenes F-111 / 李韋佑撰 - [高雄市] : 撰者, 2015[民104]. - 64面 ; 圖，表 ; 30公分.
104年10月31日公開參考書目：面44-50.
分子伴護Chaperone

偽產鹼假單胞菌F-111脂解酶之分子伴護基因選殖及特性研究 = Gene cloning and characterization of lipase chaperone from Pseudomonas pseudoalcaligenes F-111
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328 $a 碩士論文--國立高雄大學生命科學系碩士班
330 $a 大腸桿菌(Escherichia coli)在現代生物工程中經常被用作基因選殖以及生產目標蛋白質的宿主。然而,由於格蘭氏陰性菌細胞膜的內、外膜雙層結構,致使目標蛋白質往往會堆積於雙層膜之間的周質(periplasm)空間,形成不具生物活性的包涵體(inclusion body)。本篇研究期望能以Pseudomonas pseudoalcaligenes F-111菌株的脂解酶分子伴護,來研究其對於F-111脂解酶能否分泌至細胞外所扮演的角色。以PCR技術將F-111的脂解酶以及脂解酶分子伴護基因片段選殖到載體pGEM-3zf(+),電腦軟體預測顯示所選殖的脂解酶基因具備有胞外分泌所需的信號肽；並以同源模擬的方式預測所選殖的脂解酶分子伴護基因的蛋白質結構。將目標基因以E. coli表現系統進行蛋白質表現之後發現,具備完整脂解酶以及脂解酶分子伴護基因片段的重組質體能表現出脂解酶活性;然而,若將此重組質體上的脂解酶分子伴護基因剔除,儘管SDS-PAGE證實仍有脂解酶分子的表現,但是在進行活性分析時卻無法如原先的實驗結果觀察測得脂解酶活性。綜合實驗結果,研究所選殖出的脂解酶分子伴護基因,對於脂解酶的活性確實有著決定性的影響。期望此項發現能於未來建構帶有此段分子伴護基因之DNA表現載體,使大腸桿菌表現系統原本目標蛋白容易形成包涵體的問題,能在脂解酶分子伴護的協助下,便能順利送出細胞外,以增進蛋白質純化的方便性。 Escherichia coli is a commonly used gene transformation and protein production host in modern bioengineering. However, its Gram-negative double layer membrane structure also causes target protein aggregation in its periplasm space, leading to a result of inactive inclusion body formation. The aim of this study is to utilize a bacterial strain Pseudomonas pseudoalcaligenes F-111 to study its lipase chaperone's characteristics of assisting lipase secretion.By utilizing PCR, both the lipase and lipase chaperone genes from F-111 were cloned and ligated with vector pGEM-3zf(+). The computer bioinformatics prediction showed the lipase polypeptide contains a signal peptide, a necessary for secretion. Meanwhile, the structure of lipase chaperone was predicted by homology modeling. Protein analysis showed the E. coli strain transformed with complete lipase and lipase chaperone genes express lipase activities. However, if the lipase chaperone gene is knocked out, despite SDS-PAGE still shows lipase expression, it did not express its activity as the E. coli strain transformed with complete lipase and lipase chaperone genes. In conclusion, the lipase chaperone gene cloned in this study plays a crucial role in lipase activity. The study expects the discovery would lead to constructing an expression vector with the lipase chaperone gene attached for it assisting target protein secreting, to improve the convenience of protein purification in future.
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