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Immobilized β-cyclodextrin polymer coupled to agarose gel properly refolding recombinant Staphylococcus aureus elongation factor-G in combination with detergent micelle
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Chemistry, Department of Physical and Analytical Chemistry, Surface Biotechnology.
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Chemistry, Department of Physical and Analytical Chemistry, Surface Biotechnology.
Surface Biotechnology. Chemistry, Department of Physical and Analytical Chemistry, Surface Biotechnology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Physical and Analytical Chemistry.
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2006 (English)In: Protein Expression and Purification, Vol. 45, no 1, 72-79 p.Article in journal (Refereed) Published
Abstract [en]

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.
Keyword [en]
Protein refolding, Chaperone, EF-G; β-Cyclodextrin polymer, Immobilization
Identifiers
URN: urn:nbn:se:uu:diva-77526OAI: oai:DiVA.org:uu-77526DiVA: diva2:105438
Available from: 2007-02-13 Created: 2007-02-13 Last updated: 2011-01-11

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Sanyal, SuparnaJanson, Jan-Christer

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