Convenient and efficient in vitro folding of disulfide-containing globular protein from crude bacterial inclusion bodies

Junichiro Futami, Yoshiaki Tsushima, Hiroko Tada, Masaharu Seno, Hidenori Yamada

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

We investigated how the folding yield of disulfide-containing globular proteins having positive net charges from crude bacterial inclusion bodies was affected by additives in the folding buffer. In screening folding conditions for human ribonucleases and its derivative, we found that addition of salt (about 0.4 M) to a folding buffer increased the folding yield. This suggested that electrostatic interaction between polyanionic impurities such as nucleic acids and cationic unfolded protein led to the formation of aggregates under the low-salt conditions. Since inclusion bodies were found to contain nucleic acids regardless of the electrostatic nature of the expressed protein, the electrostatic interaction between phosphate moieties of nucleic acids and basic amino acid residues of a denatured protein may be large enough to cause aggregation, and therefore the addition of salt in a folding buffer may generally be useful for promotion of protein folding from crude inclusion bodies. We further systematically investigated additives such as glycerol, guanidium chloride, and urea that are known to act as chemical chaperons, and found that these additives, together with salt, synergistically improved folding yield. This study, suggesting that the addition of salt into the folding buffer is one of the crucial points to be considered, may pave the way for a systematic investigation of the folding conditions of disulfide-containing foreign proteins from crude bacterial inclusion bodies.

Original languageEnglish
Pages (from-to)435-441
Number of pages7
JournalJournal of biochemistry
Volume127
Issue number3
DOIs
Publication statusPublished - Jan 1 2000

Keywords

  • Chemical chaperon
  • Electrostatic interaction
  • Folding
  • Inclusion body
  • RNase

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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