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为了提高重组人干细胞因子(rhSCF)的复性效率,改进了高效疏水相互作用色谱(HPHIC)纯化和复性rhSCF的方法。首先将目标蛋白溶解于8.0mol/L脲中,然后将rhSCF包涵体的提取液直接进样到不同规格的HPHIC柱进行纯化和复性。优化了固定相配基结构和流动相组成等实验条件,结果表明,本方法可以快速地获得高质量回收率和高生物活性的rhSCF,rhSCF在40min内即可完成复性与纯化,目标蛋白的纯度在95.5%以上,质量回收率高于49.6%。通过体积排阻色谱和基质辅助激光解吸离子化飞行时间质谱(MALDI-TOF-MS)的分析,确认rh-SCF以单体存在。结果进一步证明HPHIC法是同时复性和纯化重组蛋白的有效工具。
In order to improve the renaturation efficiency of recombinant human stem cell factor (rhSCF), the method of purification and renaturation of rhSCF by High Performance Hydrophobic Interaction Chromatography (HPHIC) was improved. First, the target protein was dissolved in 8.0mol / L urea, then the rhSCF inclusion body extract directly into different specifications HPHIC column for purification and refolding. The experimental conditions of immobilized ligand structure and mobile phase composition were optimized. The results showed that rhSCF with high quality recovery and high biological activity could be rapidly obtained by this method. RhSCF could be refolded and purified within 40 min. Purity of target protein Above 95.5%, the quality recovery rate is higher than 49.6%. Analysis by size exclusion chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) confirmed the presence of rh-SCF as a monomer. The results further prove that the HPHIC method is an effective tool for the simultaneous renaturation and purification of recombinant proteins.