Immobilized beta-cyclodextrin polymer coupled to agarose gel properly refolding recombinant Staphylococcus aureus elongation factor-G in combination with detergent micelle.
A novel artificial chaperone system using a combination of interactions between the unfolded protein, a detergent and a chromatographic column packed with immobilized beta-cyclodextrin (beta-CD) polymer coupled to an agarose gel, was introduced to refold recombinant Staphylococcus aureus elongation factor-G (EF-G). Pre-mixing of 10% Triton X-100 and unfolded EF-G at 24 mg/ml followed by a 20-fold dilution into refolding buffer led to successful capturing of EF-G by Triton X-100 resulting in formation of a detergent-protein complex at 1.2mg/ml of final protein concentration. The complex was subsequently applied to the immobilized beta-CD polymer column resulting in correct refolding of EF-G at a concentration of 530 microg/ml with 99% mass recovery. Detergent concentrations above critical micelle concentration were required for efficient capturing of EF-G at high protein concentration. Other detergents with hydrophile-lipophile-Balance values similar to that of Triton X-100 (Triton N-101, Noindet P40 (NP40), and Berol 185) also produced similar result. Soluble polymerized beta-CD was more efficient than the monomer to remove the detergent from the protein complex in a batch system. Immobilized beta-CD polymer column further improved the capability of detergent removal and was able to prevent aggregation that occurred with the addition of soluble beta-CD polymer at high protein concentration in the batch system. The mechanism for this system-assisted refolding was tentatively interpreted: the released protein could correctly refold in an enclosed hydrophilic environment provided by the integration of matrix and beta-CD polymer, and thus avoided aggregation during detergent removal.
Li
Jing-Jing
JJ
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, China.
Venkataramana
Musturi
M
Sanyal
Suparna
S
Janson
Jan-Christer
JC
Su
Zhi-Guo
ZG
D004587:Electrophoresis, Agar Gel / Q000379:methods; D008823:Micelles; D017830:Octoxynol / Q000737:chemistry; D020653:Peptide Elongation Factor G / Q000737:chemistry*; D011108:Polymers / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D012685:Sepharose / Q000737:chemistry*; D013211:Staphylococcus aureus / Q000737:chemistry*; D013997:Time Factors; D047392:beta-Cyclodextrins / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification
D008823:Micelles; D020653:Peptide Elongation Factor G; D011108:Polymers; D011994:Recombinant Proteins; D047392:beta-Cyclodextrins; D017830:Octoxynol; D012685:Sepharose