A mild hydrophobic interaction chromatography involving polyethylene glycol immobilized to agarose media refolding recombinant Staphylococcus aureus elongation factor G
2005 (English)In: Protein Expression and Purification, Vol. 40, no 2, 327-335 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2005. Vol. 40, no 2, 327-335 p.
Elongation factor G; Refolding, Immobilized polyethylene glycol, Hydrophobic interaction chromatography, Pulse elution, Urea
IdentifiersURN: urn:nbn:se:uu:diva-70577OAI: oai:DiVA.org:uu-70577DiVA: diva2:98488