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Title: 產黃纖維單胞桿菌(Cellulomonas flavigena NTOU1)幾丁質酶C.f NTOU1 Chi50重組蛋白質新碳端標記對其親和性層析純化的影響
Authors: Wu, Tsung-Hsun
Contributors: 國立臺灣海洋大學:生命科學暨生物科技學系
Keywords: 產黃纖維單胞桿菌;幾丁質酶衍生分子;親和性純化標記
Cellulomonas flavigena;Chitinases;Affinity Purification Tags
Date: 2017
Issue Date: 2018-08-22T06:31:20Z
Abstract: 摘要 產黃纖維單胞桿菌 (Cellulomonas flavigena NTOU1) 幾丁質酶全長分子 C.f NTOU1 Chi63 之衍生分子為 C.f NTOU1 Chi50 和 C.f NTOU1 Chi38。此三個幾丁質酶分子均經前人研究並在大腸桿菌Rosetta(DE3)pLysS的表現系統中表現出具有幾丁質酶活性的重組酵素,但所表現的蛋白質量少且以 His-tag 親和性層析純化重組蛋白質效果不佳,無法完成其酵素動力學研究。 為解決此困難,擬用 PCR 方法於C.f NTOU1 Chi50重組蛋白質C端改為雙 His-tag (twin His tag)及原重組蛋白質C端 Strep tag與目標基因間增加兩個胺基酸 (serine、alanine) 作為緩衝 (linker),以期待有更好的蛋白質表現與純化效果,來完成酵素動力學的研究。目前結果顯示具雙His-tag的目標基因在HI-Control BL21(DE3)細菌宿主有較佳的蛋白質表現及親和性層析純化。 關鍵詞:產黃纖維單胞桿菌、幾丁質酶衍生分子、親和性純化標記
Abstract The full-length chitinase (C.f NTOU1 Chi63), its two protease cleavage derivatives with chitinase activity (C.f NTOU1 Chi50 and C.f NTOU1 Chi38) were cloned from Cellulomonas flavigena NTOU1. These three recombinant chitinases with either 6x His tag or Strep-6x His tag were expressed from E.coli Rosetta(DE3)pLysS with low yield and poor affinity purification efficiency. Their enzymes kinetic studies were therefore hindered to proceed. In order to solve these problems, new C-terminal tags with twin His-tag or Strep-His tag with serine/alanine linker in-between tags and target gene (C.f NTOU1 Chi50) were constructed by PCR. Hopefully, its protein expression and affinity purification efficiency will be improved and enzyme kinetics will be continued and finished. The current results showed that the target gene with new C-terminal tag of twin His-tag under the HI-Control BL21(DE3) E.coli expression host system had an obvious improvement on both protein expression level and affinity purification efficiency. Keywords:Cellulomonas flavigena; Chitinases; Affinity Purification Tags
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G001043B022.id
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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