Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3208
標題: 培養條件對內含體性質的影響
Effect of culture conditions on the characteristics of inclusion bodies
作者: 王俊幃
Wang, Jun-Wei
關鍵字: 內含體;inclusion bodies;培養條件;復性;N-acetyl-D-glucosamine-2-epimerase
出版社: 化學工程學系所
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摘要: 
利用大腸桿菌生產N-乙醯胺-D-葡萄糖-2-差向異構酶時,會形成大量的內含體,約佔重組蛋白的85%,因此內含體的性質是很重要的,為了能夠找出在不同的培養條件下產生的內含體能夠經由較簡易的步驟獲得具有活性的蛋白,因此先使用non-denatured buffer 250mM Tris buffer來回收具有活性的蛋白。
不能利用250mM Tris buffer回收的活性蛋白,便利用變性劑來獲得較多的變性蛋白之後進行後續復性的動作。在誘導培養為25℃下,用8M的Urea可以溶出總蛋白量濃度6.90mg/mL,而Gdn-HCl只可溶出總蛋白量濃度3.12mg/mL,在復性的過程中,用Urea去變性的,其可獲得的可溶蛋白回收率較高,25℃所產之內含體使用Urea和Gdn-HCl去變性,復性後其可溶蛋白回收率分別為77 %和14%。
比活性方面用Gdn-HCl來進行復性所得的比活性較高,誘導時間8小時,誘導溫度25℃,IPTG濃度為0.02mM時其比活性較其他培養條件高,分別是0.018(U/mg),0.028(U/mg),0.013(U/mg),故較佳的培養條件為誘導時間8小時,誘導溫度25℃,IPTG濃度為0.02mM。

Use Escherichia coli as a host cell to produce recombinant protein N-acetyl-D-glucosamine-2-epimerase, GlcNAc 2-epimerase. It will form large amount of incusion bodies.Therefore, it’s important to know characteristics of inclusion bodies. In order to find out what culture conditions can let us easily recovery active protein from inclusion bodies by solubilization with non-denaturing buffers, therefore, we use 250mM Tris buffer to solute it.
The active protein which can’t be recovered will be dissolved in denatured buffer it. We use 8M urea or 6M Gdn-HCl and compare the concentration of soluble protein.For the inclusion bodies formed in the temperature of 25℃, we can get 6.90mg/mL of protein concentration with Urea, but can only get 3.12mg/mL of protein concentration with Gdn-HCl.
The recovery of protein in the refolding process will be 77.04% and 14.03% in the Urea and Gdn-HCl, respectively.Gdn-HCl can produce a higher specific activity of protein in comparison with Urea.
A higher specific activity can be obtained with the culture conditions of 25℃, 0.02mM IPTG and 8 hours induction.
URI: http://hdl.handle.net/11455/3208
其他識別: U0005-2108201310511700
Appears in Collections:化學工程學系所

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