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›› 2012, Vol. 12 ›› Issue (2): 277-282.

• 生化工程专栏 • 上一篇    下一篇

亲和层析介质上蛋白质偶联位点的控制方法

冯静 黄孟军 张贵锋 孔英俊 高建萍 黄永东 苏志国   

  1. 北京中医药大学中药学院 北京中医药大学中药学院 中国科学院过程工程研究所生化工程国家重点实验室 中国科学院过程工程研究所生化工程国家重点实验室 中国科学院过程工程研究所 中国科学院过程工程研究所生化工程国家重点实验室 中国科学院过程工程研究所生化工程国家重点实验室
  • 收稿日期:2012-02-13 修回日期:2012-03-20 出版日期:2012-04-20 发布日期:2012-04-20
  • 通讯作者: 冯静

A New Method to Control the Coupling Site of Protein Ligands and Affinity Chromatographic Media

FENG Jing HUANG Meng-jun ZHANG Gui-feng KONG Ying-jun Gao Jian-Ping HUANG Yong-dong, SU Zhi-guo   

  1. School of Chinese materia medica, Beijing University of Chinese Medicine State Key laboratory of Biochemical Engineering, Institute of Process Engineering, CAS Key Laboratory of Biochemical Engineering，Institute of Process Engineering，Chinese Academy of Sciences State Key laboratory of Biochemical Engineering Institute of Process Engineering, CAS State Key laboratory of Biochemical Engineering, Institute of Process Engineering, CAS State Key Lab. Biochem. Eng., Inst. Process Eng., Chinese Academy of Sciences
  • Received:2012-02-13 Revised:2012-03-20 Online:2012-04-20 Published:2012-04-20
  • Contact: FENG Jing

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摘要： 以溶菌酶为模型蛋白，将其偶联在环氧活化的琼脂糖介质上，采用液质联用技术结合蛋白质酶切技术对琼脂糖介质表面配基蛋白的偶联位点进行了识别，考察了活化时间、偶联时间和溶液pH值对偶联位点的影响. 结果表明，环氧基密度为11.34 mmol/g、反应pH 9.5条件下，溶菌酶偶联位点发生在K96，延长偶联反应时间蛋白配基偶联量增加，对偶联位点种类无显著影响；增加环氧基密度或提高偶联反应pH值使溶菌酶通过多种位点偶联，在pH≥10.5条件下偶联位点主要发生在K33, K96和K97.

关键词: 亲和层析介质, 蛋白质配基, 偶联位点, 质谱分析, 蛋白质酶解

Abstract: Lysozyme was coupled on Sepharose 4FF media activated with 1,4-butanediol diglycidyl ether. HPLC-MS and enzymatic digestion were used to identify the coupling sites of lysozyme on the media. The effects of epoxy group density and the coupling reaction condition on the coupling sites were investigated. It was found that K96 was the main coupling site on the media when the epoxy group density was 11.34 mmol/g and pH 9.5. The coupling reaction time did not affect the coupling site. Epoxy group density and pH value during the coupling reaction are the main factors affecting the coupling sites of lysozyme on the media. More lysozyme molecules were coupled on the media via K33 and K97 when pH value was above 10.5.

Key words: affinity chromatographic media, protein ligand, coupling site, MS analysis, enzymatic digestion

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