摘要
【目的】建立聚乙二醇修饰蛋白的快速分离纯化方法。【方法】以4种聚乙二醇化药用蛋白为研究对象,采用阳离子交换树脂进行纯化。纯化条件：Hiprep 16/10 CM Sepharose FF（1 mL）预装柱;流动相：A液为20mmol/L pH4.0的醋酸缓冲液;B液为A液中加入1 mol/L NaCl;B液以0-100%离子强度线性梯度洗脱100 min,流速0.2 mL/min,波长280 nm检测,收集各洗脱峰。【结果】一步纯化可将单点修饰产物与聚乙二醇、多点修饰产物以及未修饰蛋白分离开来,单点修饰产物经SDS-PAGE检测呈单一条带,经质谱检测其相对分子质量与预期相符。【结论】所建立的纯化方法可快速分离纯化聚乙二醇修饰蛋白。
[Objective] The study established a rapid isolation and purification method for pegylated proteins. [Method] To purify 4 kinds of pegylated pharmaceutical proteins, cation ion exchange chroma- tography was used. The purification was carried out by using a Hiprep 16/10 CM Sepharose FF （1 mL） column with the mixture of （A） 20 mmol/L pH4.0 sodium acetate buffer and （B） 20 mmol/L pH4.0 sodium acetate buffer containing 1 mol/L NaCl as mobile phase in gradient mode. B buffer was eluted from 0- 100% in 100 min,at a flow rate of 0.2 mL/min and the detection wavelength was 280 nm. The eluates were collected and assayed by SDS-PAGE. [Result] The purified monopegylated proteins were achieved from the modified mixture on cation-exchange chromatography by one-step purification procedure. The monopegy- lated proteins showed single band on SDS-PAGE gel. The molecular weights of 4 kinds of monopegylated proteins were determined by MALDI-TOF mass spectrometry and the results were just as expected. [Conclusion] This developed method was suitable for rapid purification of pegylated proteins.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2009年第4期191-196,共6页
Journal of Northwest A&F University(Natural Science Edition)
基金
上海市科委重点科技攻关项目(034319217)
上海-SK研究与发展基金项目(2003002-S)
关键词
聚乙二醇
药用蛋白
分离与纯化
poly（ethylene glycol）
pharmaceutical protein
isolation and purification
