色谱

色谱

• 研究论文 • 上一篇    下一篇

高效疏水相互作用色谱法复性与同时纯化重组人Flt3配体的包涵体蛋白质

贾佳, 王骊丽*, 高栋, 耿信笃   

  1. 合成及天然功能性分子化学教育部重点实验室, 西北大学现代分离科学研究所, 陕西省分离科学重点实验室, 陕西 西安 710069
  • 收稿日期:2009-12-27 修回日期:2010-02-27 出版日期:2010-06-28 发布日期:1980-06-25
  • 通讯作者: 王骊丽

Renaturation with simultaneous purification of the recombinant human Flt3 ligand from inclusion bodies by high performance hydrophobic interaction chromatography

JIA Jia, WANG Lili*, GAO Dong, GENG Xindu   

  1. Key Laboratory of Synthetic and Natural Functional Molecular Chemistry of Ministry of Education, Institute of Modern Separation Science, Northwest University, Shaanxi Key Laboratory of Separation Science, Xi’an 710069, China
  • Received:2009-12-27 Revised:2010-02-27 Online:2010-06-28 Published:1980-06-25
  • Contact: WANG Lili

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642

被引次数

8

摘要： Flt3配体(FL)是一类具有促进早期造血功能的细胞因子,在促进造血细胞生长发育及造血动员方面具有重要的临床应用价值。为了用基因工程方法获得大量用于临床和研究的重组人FL(rhFL)蛋白质,本文对在大肠杆菌(E. coli)中表达得到的Flt3配体的包涵体进行回收、洗涤,溶解于8 mol/L脲后在高效疏水相互作用色谱(HPHIC)柱上进行rhFL包涵体的复性与同时纯化,并对其保留特征和复性规律进行了研究。结果表明,在连续进样、变性蛋白质质量浓度为8.51 g/L、固定相选用端基为PEG800、流动相添加4 mol/L脲、1.8 mmol/L 还原型谷胱甘肽(GSH)和0.3 mmol/L氧化型谷胱甘肽(GSSH)、pH 7.0的优化条件下,复性与同时纯化rhFL包涵体的质量回收率为36.9%,纯度达94.5%以上。本文仅用一步HPHIC法成功地复性与同时纯化了rhFL蛋白质,为获得高活性的rhFL产品奠定了一定的工作基础。

关键词: 包涵体 , 纯化, 复性, 高效疏水相互作用色谱法, 重组人Flt3配体

Abstract: Flt3 ligand (FL) is a class of cytokines with the functions of promoting early hematopoiesis. It has important clinical value in promoting growth and development of hematopoietic cells and hematopoietic mobilization. In order to obtain large quantities of recombinant human FL (rhFL) by genetic engineering methods for clinic and research, in this work, rhFL was expressed in E. coli as inclusion bodies. The inclusion bodies were recovered, cleaned and solubilized in 8 mol/L urea, the solubilized rhFL was renatured by high performance hydrophobic interaction chromatography (HPHIC) with simultaneous purification, the retention feature and renaturation regularity were studied. The results showed that when the denatured protein concentration was 8.51 g/L, and the end group of stationary phase was PEG800, under the conditions of mobile phase of pH 7.0 and with the addition of 4 mol/L urea, 1.8 mmol/L glutathione (GSH) and 0.3 mmol/L oxidative glutathione (GSSG), a mass recovery of 36.9% and a purity of 94.5% were obtained after refolding with simultaneous purification. The obtained rhFL was successfully renatured with simultaneous purification in only one step of HPHIC, and it provided a foundation for the manufacturing of high quality rhFL.

Key words: inclusion bodies , purification, recombinant human Fms-like tyrosine kinase ligand 3 (rhFL), renaturation, high performance hydrophobic interaction chromatography (HPHIC)