重组大肠杆菌表达的肠激酶的纯化工艺优化研究
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