蛋白折叠液相色谱法中流动相对重组人干扰素-γ质量回收率的影响  

Effect of Mobile Phase on the Mass Recovery of rhIFN-γ in Protein Folding Liquid Chromatography

作　　者：吴丹[1] 王超展[2] 耿信笃[2] 

机构地区：[1]陕西西大科林基因药业有限公司,陕西西安710075 [2]西北大学现代分离科学研究所现代分离科学陕西省重点实验室,陕西西安710069

出　　处：《色谱》2007年第2期197-202,共6页Chinese Journal of Chromatography

基　　金：国家自然科学基金资助项目(No.20175016;20475042)

摘　　要：蛋白折叠液相色谱法(PFLC)用于变性蛋白质复性并同时纯化时对流动相组成及其洗脱条件的要求远较通常的液相色谱法高。用端基为PEG-200的高效疏水作用色谱固定相对重组人干扰素-γ(rhIFN-γ)进行纯化并同时复性,详细研究了流动相组成、梯度洗脱模式和流速对rhIFN-γ质量回收率和活性的影响。分别以3.0mol/L(NH4)2SO4+0.05mol/L KH2PO4(pH7.0)和0.05mol/L KH2PO4(pH7.0)为流动相A和B,采用35min非线性梯度洗脱时,所得rhIFN-γ的质量回收率最高。Protein refolding is a key process for the production of recombinant proteins from E. coli. One of the effective techniques for protein refolding is through liquid chromatography named as protein folding liquid chromatography （PFLC）. Mobile phase composition is one of the most important factors affecting the efficiency of protein renaturation in PFLC. Optimization of mobile phase composition in PFLC for protein renaturation with simultaneous purification is thus much more important than that in usual liquid chromatography. This study presents an approach with regard to the increase of protein production through optimizing the mobile phase composition of PFLC. Recombinant human interferon-γ （rhIFN-γ ） was purified with simultaneous renaturation by high performance hydrophobic interaction chromatography （ HPHIC） using a stationary phase with an end group of PEG-200. Effects of mobile phase composition, gradient elution mode, and flow rate on the recoveries of mass and bioactivity of rhIFN-γ were investigated. It was found that these factors were very important for refolding with simultaneous purification of rhIFN-γ by PFLC. This study highlights the importance of optimizing the mobile phase composition in PFLC for increasing the recovery of protein. Taking 3.0 moVL （NH4）2SO4 ＋0. 05 mol/L KH2PO4 （pH 7.0） and 0.05 mol/L KH2PO4 （pH 7.0） as mobile phases A and B, respectively, the highest mass recovery was obtained under a nonlinear gradient elution for 35 min.

关 键 词：高效疏水作用色谱法 流动相组成 梯度洗脱模式 纯化 复性 质量回收率 重组人干扰素-γ 

分 类 号：O658[理学—分析化学]