The Optimization of the Recombinant Molecular Weight Standard Protein Mass Production in Escherichia Coli and Its Staining

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基本資料

Project title

Code/計畫編號

MOST109-2622-B019-003-CC2

Translated Name/計畫中文名

重組分子量標準蛋白的量產與其染色之最適化探討

Project Coordinator/計畫主持人

Tsuei-Yun Fang

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Food Science

Website

https://www.grb.gov.tw/search/planDetail?id=13443692

Year

2020

Start date/計畫起

01-06-2020

Expected Completion/計畫迄

31-05-2021

Bugetid/研究經費

1000千元

ResearchField/研究領域

食品科技(農)

任何蛋白質相關研究皆須使用分子量標準蛋白，而經預先染色而製成之預染蛋白標準試劑(prestained protein standards)，更是西方點墨法中不可或缺的試劑。本計畫將利用107及108年科技部的產學計畫所獲得之12個分別可表現分子量10~200 kDa (10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 kDa)重組蛋白的E. coli BL21 Star (DE3)或BL21(DE3)-CodonPlus-RIL最佳重組菌株，利用先前使用搖瓶所獲得之最適的培養與誘導條件，於發酵槽以饋料批式發酵方式進行高細胞密度培養，以大量生產可表現分子量10~200 kDa重組蛋白的大腸桿菌菌體，並且探討饋料添加方式對於10~200 kDa重組蛋白生產量與菌體收穫量的影響;在其回收純化的部分，將純化管柱由原本5毫升膠體放大至40 ml ，比較管柱膠體體積及所使用imidazole濃度梯度，對於重組蛋白純化後的純度、濃度及產量之影響，找出最佳條件以減低生產成本;針對染色最適化的部分，將比較緩衝液種類與pH值、染料濃度、染料顏色(紅綠藍3種顏色)、反應溫度與時間，對於各個重組蛋白質染色的效率及色帶清晰度的影響; 最終將協助廠商建立由養菌至染色成品的完整量產流程。本計畫主要目的是藉由重組分子量標準蛋白的量產與其染色之最適化探討，提高由單位體積培養液中純化所得之重組蛋白產量，並且提升蛋白質染色的效率及色帶清晰度，降低未染色及預染蛋白標準試劑的生產成本，能為台灣創造商機，最終有助於蛋白質相關研究的發展。 Every protein related investigation must use protein standards, and the prestained protein standards are the crucial reagents in western blotting. This proposal will use 12 recombinant Escherichia coli BL21 Star (DE3) or BL21(DE3)-CodonPlus-RIL stains which obtained from Industry-Academia Cooperation Project of Ministry of Science and Technology in the years of 107 and 108. These strains can express recombinant proteins with molecular weights of 10~200 kDa (10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, and 200 kDa), respectively. The proper culturing and induction conditions from growing in shaking flasks will be applied in feed batch cultivation using stirred tank fermenter to achieve high cell density in order to mass produce the recombinant proteins with molecular weights of 10~200 kDa. We will investigate the effect of feeding-strategy of fed-batch fermentation on the production of recombinant proteins and cell mass. In the recovery and purification, the resins packed in the column of affinity chromatography will be enlarged from 5 mL to 40 mL, and the effects of resin volumes and imidazole concentration gradients on the purity, concentration, and yield of the recombinant will be compared. As to staining optimization, the effects of buffer type, pH value, dye concentration, dye color, reaction temperature, and reaction time on the staining efficiency and and band sharpness will be compared. Finally, we will assist the cooperated corporation to establish a complete mass production process from the growing of the bacterial culture to obtain prestained protein standards productsThis proposal aims to optimize the production of recombinant molecular weight standard proteins and their staining conditions. We wish to increase the yield of recombinant protein purified from per unit volume of culture medium, and to improve the efficiency of protein staining and the band sharpness, and to reduce the production costs of both unstained and prestained proteins standards. This can create business opportunities for Taiwan and ultimately help the development of protein-related research.

Keyword(s)

大腸桿菌
重組蛋白
饋料批式發酵
預染蛋白標準試劑
Escherichia coli
recombinant protein
fed-batch fermentation
prestained protein standards

DSpace CRIS

The Optimization of the Recombinant Molecular Weight Standard Protein Mass Production in Escherichia Coli and Its Staining

基本資料

Description