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 beta-cyclodextrin (beta-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 beta-CD concentration gradient. The yield of properly refolded GFPTEVP was 46% at a protein concentration of 380 mu g/ml. In contrast, dilution refolding of GFPTEVP at 200 mu g/ml refolding buffer resulted in only 4.7% of native protein. (C) 2008 Elsevier Ltd. All rights reserved.