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重組大腸桿菌表達的腸激酶的純化工藝優化研究

時間:2019-04-13 14:34來源:畢業論文
腸激酶存在于高等動物的十二指腸粘膜中,是使胰蛋白酶原水解而成為胰蛋白酶的肽鏈內切酶。重組大腸桿菌高密度表達腸激酶輕鏈融合蛋白Trx-rEKL,主要以包涵體形式存在[1],通過純化

摘要:【目的】腸激酶存在于高等動物的十二指腸粘膜中,是使胰蛋白酶原水解而成為胰蛋白酶的肽鏈內切酶。重組大腸桿菌高密度表達腸激酶輕鏈融合蛋白Trx-rEKL,主要以包涵體形式存在[1],通過純化工藝優化研究來制備腸激酶的粗酶液。【實驗】發酵結束后,離心收集菌體,按重量體積比 1∶8溶于TE緩沖液, 高壓均質機900 bar、40 hz條件下破碎兩遍,4000 r/min、30 min離心收集包涵體沉淀,將沉淀用TE緩沖液徹底清洗,再次離心收集沉淀并稱得包涵體濕重,根據相應比例加水配成包涵體水溶液,攪拌器攪拌均勻后,加入urea、0.5 mol EDTA、20 mmol DTT變性過夜,變性后的包涵體經750 K柱超濾去除雜質后,利用分光光度法測出蛋白濃度,根據復性緩沖液終濃度<0.3 mg/ml的標準,在純化水中加入氨基乙酸、谷胱甘肽(還原型)、谷胱甘肽(氧化型)配成相應體積復性緩沖液,以脈沖形式加樣進復性緩沖液中,4 ℃復性過夜,復性樣品用3 K柱超濾濃縮后得到的腸激酶原蛋白經腸激酶(1:200)在室溫下酶切過夜后即可得到具有活性的粗酶液[2]。【結果】粗酶液使用GenericMC-SP陽離子柱層析分離即可獲得較純的腸激酶樣品。34462
畢業論文關鍵詞:腸激酶;包涵體;變性;復性;純化
Study on the Purification Process Optimization of Recombinant Escherichia coli Expression of Enterokinase
SONG Jingwei      Supervisor: Peng Xue
(School of Life Sciences, Jiangsu Normal University, Xuzhou, Jiangsu, 221116, China)
Abstract:【Purpose】 The intestinal kinase exists in the duodenal mucosa of higher animals,it is an endopeptidase which can make trypsinogen into trypsase. The high expression of recombinant Escherichia coli eklc fusion protein Trx-rEKL,mainly in   源¥自%六^^維*論-文+網=www.aftnzs.live
the form of inclusion body[1]. 【Experiment】After the end of fermentation, centrifugal bacteria were collected by volume weight ratio of 1: 8 dissolved in the buffer and high pressure homogenizer 900 bar, 40 Hz condition broken twice, 4000 r / min, 30 min centrifugal collected inclusion precipitation, precipitation will wash thoroughly with the buffer, and again collected by centrifugation sedimentation and called inclusion body wet weight,according to corresponding proportion of water with inclusion aqueous solution, stirring evenly, adding urea and 0.5 mol EDTA, 20 mmol DTT degeneration of the overnight,Denatured inclusion bodies by 750 k hyperfiltration column to remove impurities, using spectrophotometric method to measure the concentration of protein, according to the complex buffer final concentration <0.3 mg/ml standard and in purified water adding amino acetic acid, glutathione (GSH), glutathione (GSSG) with corresponding volume of refolding buffer,to pulse form and rehabilitation into the buffer, 4 ℃ renaturation overnight, refolded sample with 3K column after ultrafiltration by enterokinase protein by enterokinase (1:200) at room temperature enzyme digestion after overnight can be obtained with activity of crude enzyme solution[2].【Consequence】The crude enzyme solution using GenericMC-SP cation column chromatography to obtain pure enterokinase samples.                          
Key words:Enterokinase; inclusion; denaturation; renaturation; purification
目錄
引言    1
1材料與方法    2
1.1供試材料    2
1.1.1質粒和菌株    2
1.1.2儀器和設備    2
1.1.3試劑    2
1.2方法    2
1.2.1重組大腸桿菌的發酵    2 源¥自%六^^維*論-文+網=www.aftnzs.live
1.2.2重組大腸桿菌的破碎    2
1.2.3包涵體的變性    3
1.2.4變性液的超濾    3 重組大腸桿菌表達的腸激酶的純化工藝優化研究:http://www.aftnzs.live/shengwu/20190413/31968.html
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