重组大肠杆菌表达的肠激酶的纯化工艺优化研究
时间:2019-04-13 14:34 来源:毕业论文 作者:毕业论文 点击:次
| 摘要：【目的】肠激酶存在于高等动物的十二指肠粘膜中，是使胰蛋白酶原水解而成为胰蛋白酶的肽链内切酶。重组大肠杆菌高密度表达肠激酶轻链融合蛋白Trx-rEKL,主要以包涵体形式存在[1]，通过纯化工艺优化研究来制备肠激酶的粗酶液。【实验】发酵结束后，离心收集菌体，按重量体积比 1∶8溶于TE缓冲液, 高压均质机900 bar、40 hz条件下破碎两遍，4000 r/min、30 min离心收集包涵体沉淀，将沉淀用TE缓冲液彻底清洗，再次离心收集沉淀并称得包涵体湿重，根据相应比例加水配成包涵体水溶液，搅拌器搅拌均匀后，加入urea、0.5 mol EDTA、20 mmol DTT变性过夜，变性后的包涵体经750 K柱超滤去除杂质后，利用分光光度法测出蛋白浓度，根据复性缓冲液终浓度<0.3 mg/ml的标准，在纯化水中加入氨基乙酸、谷胱甘肽（还原型）、谷胱甘肽（氧化型）配成相应体积复性缓冲液，以脉冲形式加样进复性缓冲液中，4 ℃复性过夜，复性样品用3 K柱超滤浓缩后得到的肠激酶原蛋白经肠激酶（1:200）在室温下酶切过夜后即可得到具有活性的粗酶液[2]。【结果】粗酶液使用GenericMC-SP阳离子柱层析分离即可获得较纯的肠激酶样品。34462 毕业论文关键词：肠激酶;包涵体;变性;复性;纯化 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 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 目录 引言 1 1材料与方法 2 1.1供试材料 2 1.1.1质粒和菌株 2 1.1.2仪器和设备 2 1.1.3试剂 2 1.2方法 2 1.2.1重组大肠杆菌的发酵 2 1.2.2重组大肠杆菌的破碎 2 1.2.3包涵体的变性 3 1.2.4变性液的超滤 3 (责任编辑：qin) |