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首页> 外文期刊>Biotechnology Progress >High Recovery Refolding of rhG-CSF from Escherichia coli,Using Urea Gradient Size Exclusion Chromatography
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High Recovery Refolding of rhG-CSF from Escherichia coli,Using Urea Gradient Size Exclusion Chromatography

机译：使用尿素梯度大小排阻色谱法从大肠杆菌中高回收度重组rhG-CSF

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Protein folding liquid chromatography(PFLC)is a powerful tool for simultaneous refolding and purification of recombinant proteins in inclusion bodies.Urea gradient size exclusion chromatography(SEC)is a recently developed protein refolding method based on the SEC refolding principle.In the presented work,recombinant human granulocyte colony-stimulating factor(rhG-CSF)expressed in Escheridchia coli(E.coli)in the form of inclusion bodies was refolded with high yields by this method.Denatured/reduced rhG-CSF in 8.0 mol·L~(-1)urea was directly injected into a Superdex 75 column,and with the running of the linear urea concentration program,urea concentration in the mobile phase and around the denatured rhG-CSF molecules was decreased linearly,and the denatured rhG-CSF was gradually refolded into its native state.Aggregates were greatly suppressed and rhG-CSF was also partially purified during the refolding process.Effects of the length and the final urea concentration of the urea gradient on the refolding yield of rhG-CSF by using urea gradient SEC were investigated respectively.Compared with dilution refolding and normal SEC with a fixed urea concentration in the mobile phase,urea gradient SEC was more efficient for rhG-CSF refolding-in terms of specific bioactivity and mass recovery,the denatured rhG-CSF could be refolded at a larger loading volume,and the aggregates could be suppressed more efficiently.When 500 mu L of solubilized and denatured rhG-CSF in 8.0 mol·L~(-1)urea solution with a total protein concentration of 2.3 mg·mL~(-1)was loaded onto the SEC column,rhG-CSF with a specific bioactivity of 1.0 x 10~8 IU·mg~(-1)was obtained,and the mass recovery was 46.1%.
机译：蛋白质折叠液相色谱法（PFLC）是同时重组和纯化包涵体中重组蛋白质的有力工具。尿素梯度大小排阻色谱法（SEC）是基于SEC折迭原理的最新开发的蛋白质折迭方法。该方法以高产率重折叠以包涵体形式在大肠杆菌中表达的重组人粒细胞集落刺激因子（rhG-CSF）。变性/还原的rhG-CSF在8.0mol·L〜（- 1）将尿素直接注入Superdex 75色谱柱中，并随着线性尿素浓度程序的运行，流动相和变性的rhG-CSF分子周围的尿素浓度线性降低，变性的rhG-CSF逐渐重折叠在重折叠过程中，聚集体被大大抑制且rhG-CSF也被部分纯化尿素梯度o的长度和最终尿素浓度的影响在分别研究了使用尿素梯度SEC进行rhG-CSF的重折叠产率方面。与稀释重折叠和在流动相中具有固定尿素浓度的正常SEC相比，尿素梯度SEC对rhG-CSF的重折叠更有效。生物活性和质量回收率，变性的rhG-CSF可以在较大的上样量下重新折叠，并且可以更有效地抑制聚集体。当在8.0mol·L〜（-1）尿素中溶解并变性的rhG-CSF达到500μL时将总蛋白浓度为2.3 mg·mL〜（-1）的溶液上样至SEC柱上，得到比生物活性为1.0 x 10〜8 IU·mg〜（-1）的rhG-CSF，质量回收率为46.1％。

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