title: Direct refolding of inclusion bodies using reversed micelles.
abstract: The protein refolding of inclusion bodies was investigated using reversed micelles formed by aerosol OT (AOT). Ribonuclease A (RNase A) was overexpressed in Escherichia coli and used as native inclusion bodies. The enzymatic activity of RNase A was completely regained from the inclusion bodies within 14 h by solubilization in reversed micelles. To further enhance the refolding rate, a molecular chaperone, GroEL, was incorporated into the refolding system. The resultant refolding system including GroEL showed better performance under optimized conditions for the refolding of RNase A inclusion bodies. The refolding rate was considerably improved by the addition of the molecular chaperone, and the refolding step was completed in 1 h. The protein refolding in the GroEL-containing refolding system was strongly dependent on the coexistence of ATP and Mg2+, suggesting that the GroEL hosted in the reversed micelles was biologically active and assisted in the renaturation of the inclusion bodies. The addition of cold acetone to the reversed micellar solution allowed over 90% recovery of the renatured RNase A.
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 6-10-1, Hakozaki, Fukuoka 812-8581, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Sakono
authors.forename: Yu-mi
authors.initials: YM
authors.lastname: Kawashima
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Ichinose
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Maruyama
authors.forename: Noriho
authors.initials: N
authors.lastname: Kamiya
authors.forename: Masahiro
authors.initials: M
authors.lastname: Goto
mesh_terms: D000818:Animals; D002417:Cattle; D018834:Chaperonin 60 / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry* / Q000201:enzymology*; D008823:Micelles; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D018834:Chaperonin 60; D008823:Micelles; D011994:Recombinant Proteins; D012259:Ribonuclease, Pancreatic