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Immobilized Triton X-100-assisted refolding of Green Fluorescent Protein-Tobacco Etch Virus protease fusion protein using β-cyclodextrin as the eluent
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry. (Ytbioteknik)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Surface Biotechnology.
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2009 (English)In: Process Biochemistry, ISSN 1359-5113, E-ISSN 1873-3298, Vol. 44, no 3, 277-282 p.Article in journal (Refereed) Published
Abstract [en]

A new protein refolding technique based on the use of the non-charged detergent Triton X-100 immobilized to the cross-linked agarose gel Sepharose High Performance has been developed. The new solid phase was used in combination with soluble β-cyclodextrin (β-CD) to refold recombinant Green Fluorescent Protein fused to Tobacco Etch Virus protease (GFPTEVP) expressed as inclusion bodies in E. coli. Previous attempts to refold recombinant GFPTEVP by dilution had failed. In the new procedure a column packed with Triton X-100-coupled Sepharose High Performance was used to capture unfolded GFPTEVP followed by elution using an increasing β-CD concentration gradient. The yield of properly refolded GFPTEVP was 46% at a protein concentration of 380 μg/ml. In contrast, dilution refolding of GFPTEVP at 200 μg/ml refolding buffer resulted in only 4.7% of native protein.

Place, publisher, year, edition, pages
2009. Vol. 44, no 3, 277-282 p.
Keyword [en]
Immobilized Triton X-100, Sepharose High Performance, Refolding, Green Fluorescent Protein, Hydrophobic interaction chromatography, Artificial chaperone, β-Cyclodextrin
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-99052DOI: 10.1016/j.procbio.2008.10.021ISI: 000264148400004OAI: oai:DiVA.org:uu-99052DiVA: diva2:202032
Available from: 2009-03-06 Created: 2009-03-06 Last updated: 2017-12-13Bibliographically approved

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