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On‐Column Refolding of Recombinant Human Interferon‐γ with an Immobilized Chaperone Fragment
Author(s) -
Gao YongGui,
Guan YiXin,
Yao ShanJing,
Cho ManGi
Publication year - 2003
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp025775l
Subject(s) - groel , chemistry , sephadex , chaperone (clinical) , chromatography , recombinant dna , inclusion bodies , protein folding , specific activity , in vitro , biochemistry , biophysics , escherichia coli , enzyme , biology , medicine , pathology , gene
The chaperone mini‐GroEL is a soluble recombinant fragment containing the 191–345 amino acid sequence of GroEL with a 6×His tag. The refolding protocol assisted with mini‐GroEL was studied for the activity recovery of rhIFN‐γ inclusion bodies. In a suspended system, mini‐GroEL showed significant enhancement of the activity recovery of rhIFN‐γ, applyed with a 1–5:1 stoichiometry of mini‐GroEL to rhIFN‐γ at 25 °C. Moreover, 1 M urea in the renaturation buffer had a synergistic effect on suppressing the aggregation and improving the activity recovery. Finally, a novel chromatographic column, containing 1 cm height of Sephadex G 200 at the top of column and packed with immobilized mini‐GroEL to promote refolding, was devised. The total activity recovered per milligram of denatured rhIFN‐γ was up to 3.93 × 10 6 IU with the immobilized mini‐GroEL column, which was reused four times without evident loss of renaturation ability. A convenient technique with the integrated process of chaperon preparation and rhIFN‐γ folding in vitro was developed.