title: A mild hydrophobic interaction chromatography involving polyethylene glycol immobilized to agarose media refolding recombinant Staphylococcus aureus elongation factor G.
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.
authors.affiliation: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, China.
authors.forename: Jing-Jing
authors.initials: JJ
authors.lastname: Li
authors.forename: Musturi
authors.initials: M
authors.lastname: Venkataramana
authors.forename: Ai-Qing
authors.initials: AQ
authors.lastname: Wang
authors.forename: Suparna
authors.initials: S
authors.lastname: Sanyal
authors.forename: Jan-Christer
authors.initials: JC
authors.lastname: Janson
authors.forename: Zhi-Guo
authors.initials: ZG
authors.lastname: Su
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D002845:Chromatography / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D020653:Peptide Elongation Factor G / Q000737:chemistry* / Q000235:genetics; D011092:Polyethylene Glycols; D017510:Protein Folding; D011994:Recombinant Proteins; D012685:Sepharose; D013211:Staphylococcus aureus / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D020653:Peptide Elongation Factor G; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D012685:Sepharose