Immobilized β-cyclodextrin polymer coupled to agarose gel properly refolding recombinant Staphylococcus aureus elongation factor-G in combination with detergent micelle
2006 (English)In: Protein Expression and Purification, Vol. 45, no 1, 72-79 p.Article in journal (Refereed) Published
A novel artificial chaperone system using a combination of interactions between the unfolded protein, a detergent and a chromatographic column packed with immobilized β-cyclodextrin (β-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 1.2 mg/ml of final protein concentration. The complex was subsequently applied to the immobilized β-CD polymer column resulting in correct refolding of EF-G at a concentration of 530 μg/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 β-CD was more efficient than the monomer to remove the detergent from the protein complex in a batch system. Immobilized β-CD polymer column further improved the capability of detergent removal and was able to prevent aggregation that occurred with the addition of soluble β-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 β-CD polymer, and thus avoided aggregation during detergent removal.
Place, publisher, year, edition, pages
2006. Vol. 45, no 1, 72-79 p.
Protein refolding, Chaperone, EF-G; β-Cyclodextrin polymer, Immobilization
IdentifiersURN: urn:nbn:se:uu:diva-77526OAI: oai:DiVA.org:uu-77526DiVA: diva2:105438