The Optimization of the Recombinant Molecular Weight Standard Protein Expression in Escherichia Coli and Its Recovery and Purification(I)

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Project title

Code/計畫編號

MOST107-2622-B019-001-CC2

Translated Name/計畫中文名

重組分子量標準蛋白在大腸桿菌表現與其回收純化之最適化探討(1/2)

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=12587543

Year

2018

Start date/計畫起

01-06-2018

Expected Completion/計畫迄

31-05-2019

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。本計畫預定分兩年執行，由合作企業提供12個重組質體，這些質體在大腸桿菌中分別可表現出12個不同分子量的重組蛋白，是該公司欲開發之預染蛋白標準試劑的原料。於第一年核定的計畫中，先探討6個重組分子量標準蛋白 (分子量 10, 30, 50, 70, 90 及 150 kDa)在大腸桿菌表現與其回收純化之最適化，已透過適當的啓動子與宿主菌株選擇、培養與誘導條件最適化，有效提升重組蛋白表現量。也已嘗試在較低溫度或在化學伴護子存在下誘導其表現，並將重組蛋白與分子伴護蛋白共表現，以提升重組蛋白之可溶性蛋白的表現量。原先這些重組蛋白在大腸桿菌中的表現不理想，由每公升的培養液中純化所得之polyHistagged重組蛋白產量僅約有4~40 mg，透過表現與回收純化之最適化探討，在分子量30, 50, 70及 150 kDa的部分，每公升細菌培養液分別可獲得 950, 200, 150及55 mg純化蛋白，其蛋白產量已大幅提昇，並且簡化重組蛋白的萃取與純化流程。計畫第一年後期會繼續探討10, 90, 150 kDa的回收純化之最適化。本計畫目的是提高由單位體積培養液中純化所得之重組蛋白產量，降低生產成本，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. In this two-year proposal, the cooperated corporation would provide us 12 recombinant plasmids; these plasmids could be expressed in Escherichia coli to produce 12 recombinant proteins with different molecular weights. These recombinant proteins are the raw materials for developing new products of molecular weight standard proteins (prestained protein standards) in the cooperated corporation. In the first year of this proposal, we have investigated the expressions, purification and recovery of 6 recombinant proteins with molecular weights of 10, 30, 50, 70, 90, and 150 kDa, respectively. We have chosen the proper promoters and host strains, optimized culturing and induction conditions, and therefore increase the expressions of recombinant proteins. We have also tried to induce the expressions of recombinant proteins under lower temperature and in the presence of chemical chaperones, and co-expressed the recombinant proteins with molecular chaperones to enhance the expression of soluble recombinant proteins. The yields of these recombinant proteins were only 4~40 mg per liter of culture medium before optimization. After optimization, the yields of recombinant proteins of molecular weights of 30, 50, 70, and 150 kDa have achieved 950, 200, 150, and 55 mg/L, respectively. Not only the recombinant protein yields are greatly improved, the processes of extraction and purification are also simplified. In the final period of the first-year proposal, we will continually investigate the optimization of recovery and purification of recombinant proteins of molecular weights of 10, 90, and 150 kDa. This proposal aims to enhance the final yield of recombinant proteins from per unit volume of culture medium. Then, the price for producing recombinant proteins will be lowered, and finally it will benefit the developments of protein-related researches.

Keyword(s)

大腸桿菌
重組蛋白
蛋白質表現
伴護子
Escherichia coli
recombinant protein
protein expression
chaperone

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The Optimization of the Recombinant Molecular Weight Standard Protein Expression in Escherichia Coli and Its Recovery and Purification(I)

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