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 language | English |
|---|---|
| Pages (from-to) | 435-441 |
| Number of pages | 7 |
| Journal | Journal of Biochemistry |
| Volume | 127 |
| Issue number | 3 |
| Publication status | Published - 2000 |
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Keywords
- Chemical chaperon
- Electrostatic interaction
- Folding
- Inclusion body
- RNase
ASJC Scopus subject areas
- Biochemistry
Cite this
Convenient and efficient in vitro folding of disulfide-containing globular protein from crude bacterial inclusion bodies. / Futami, Junichiro; Tsushima, Yoshiaki; Tada, Hiroko; Seno, Masaharu; Yamada, Hidenori.
In: Journal of Biochemistry, Vol. 127, No. 3, 2000, p. 435-441.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Convenient and efficient in vitro folding of disulfide-containing globular protein from crude bacterial inclusion bodies
AU - Futami, Junichiro
AU - Tsushima, Yoshiaki
AU - Tada, Hiroko
AU - Seno, Masaharu
AU - Yamada, Hidenori
PY - 2000
Y1 - 2000
N2 - 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.
AB - 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.
KW - Chemical chaperon
KW - Electrostatic interaction
KW - Folding
KW - Inclusion body
KW - RNase
UR - http://www.scopus.com/inward/record.url?scp=0034072664&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0034072664&partnerID=8YFLogxK
M3 - Article
VL - 127
SP - 435
EP - 441
JO - Journal of Biochemistry
JF - Journal of Biochemistry
SN - 0021-924X
IS - 3
ER -