Structural characteristics and refolding of in vivo aggregated hyperthermophilic archaeon proteins

Mitsuo Umetsu, Kouhei Tsumoto, Kumar Ashish, Shigeki Nitta, Yoshikazu Tanaka, Tadafumi Adschiri, Izumi Kumagai

研究成果: Article査読

31 被引用数 (Scopus)

抄録

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 β-strand-rich single chain Fv fragment (scFv) andz α-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 α-helix rich, are predominated by α-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 α-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.

本文言語English
ページ(範囲)49-56
ページ数8
ジャーナルFEBS Letters
557
1-3
DOI
出版ステータスPublished - 2004 1 16

ASJC Scopus subject areas

  • 生物理学
  • 構造生物学
  • 生化学
  • 分子生物学
  • 遺伝学
  • 細胞生物学

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