On-column refolding of recombinant human interferon-gamma with an immobilized chaperone fragment.
The chaperone mini-GroEL is a soluble recombinant fragment containing the 191-345 amino acid sequence of GroEL with a 6xHis tag. The refolding protocol assisted with mini-GroEL was studied for the activity recovery of rhIFN-gamma inclusion bodies. In a suspended system, mini-GroEL showed significant enhancement of the activity recovery of rhIFN-gamma, applyed with a 1-5:1 stoichiometry of mini-GroEL to rhIFN-gamma at 25 degrees 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-gamma was up to 3.93 x 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-gamma folding in vitro was developed.
Gao
Yong-Gui
YG
Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, China.
Guan
Yi-Xin
YX
Yao
Shan-Jing
SJ
Cho
Man-Gi
MG
D000327:Adsorption; D018834:Chaperonin 60 / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry*; D007371:Interferon-gamma / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D018832:Molecular Chaperones / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D018834:Chaperonin 60; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D007371:Interferon-gamma