Related Experiment Video
Updated: Aug 10, 2026
Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification
Published on: September 21, 2011
A mild hydrophobic interaction chromatography involving polyethylene glycol immobilized to agarose media refolding
Jing-Jing Li1, Musturi Venkataramana, Ai-Qing Wang
1National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, China.
Abstract:
Recombinant Staphylococcus aureus elongation factor G (EF-G) is difficult to refold by dilution due to the formation of large amounts of misfolded structures. However, refolding of EF-G by adsorption to a chromatographic column packed with immobilized polyethylene glycol 20,000 (PEG 20 K) followed by pulse elution with 8 M urea resulted in 88% mass recovery and 80% of correctly refolded structure. The PEG 20 K was coupled to brominated allyl group derivatized Sepharose High Performance to construct a mild hydrophobic adsorbent. Various other hydrophobic interaction adsorbents were also attempted to refold EF-G. However, ligands with high hydrophobicity tended to misfold EF-G, resulting in irreversible adsorption. Various solvents, detergents, and low temperature as well as 8 M urea were tried to release bound EF-G. Only pulse elution with 8 M urea was efficient. Urea concentrations favorable for efficiently refolding EF-G were investigated. Low urea concentration produced more misfolded structures.
