title: Structural characteristics and refolding of in vivo aggregated hyperthermophilic archaeon proteins.
abstract: Several recombinant proteins in inclusion bodies expressed in Escherichia coli have been measured by Fourier transform infrared and solid-state nuclear magnetic resonance spectra to provide the secondary structural characteristics of the proteins from hyperthermophilic archaeon Pyrococcus horikoshii OT3 (hyperthermophilic proteins) in inclusion bodies. The beta-strand-rich single chain Fv fragment (scFv) and alpha-helix-rich interleukin (IL)-4 lost part of the native-like secondary structure in inclusion bodies, while the inclusion bodies composed of the hyperthermophilic proteins of which the native form is alpha-helix rich, are predominated by alpha-helix structure. Further, the secondary structure of the recombinant proteins solubilized from inclusion bodies by detergent or denaturant was observed by circular dichroism (CD) spectra. The solubilization induced the denaturation of the secondary structure for scFv and IL-4, whereas the solubilized hyperthermophilic proteins have retained the alpha-helix structure with the CD properties resembling those of their native forms. This indicates that the hyperthermophilic proteins form native-like secondary structure in inclusion bodies. Refolding of several hyperthermophilic proteins from in vivo aggregated form without complete denaturation could be accomplished by solubilization with lower concentration (e.g. 2 M) of guanidine hydrochloride and removal of the denaturant via stepwise dialysis. This supports the existence of proteins with native-like structure in inclusion bodies and suggests that non-native association between the secondary structure elements leads to in vivo aggregation. We propose a refolding procedure on the basis of the structural properties of the aggregated archaeon proteins.
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 07, Aoba-ku, Sendai 980-8579, Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Umetsu
authors.forename: Kouhei
authors.initials: K
authors.lastname: Tsumoto
authors.forename: Kumar
authors.initials: K
authors.lastname: Ashish
authors.forename: Shigeki
authors.initials: S
authors.lastname: Nitta
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Tadafumi
authors.initials: T
authors.lastname: Adschiri
authors.forename: Izumi
authors.initials: I
authors.lastname: Kumagai
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D006358:Hot Temperature; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D019711:Pyrococcus / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D019843:Archaeal Proteins