Convenient and efficient in vitro folding of disulfide-containing globular protein from crude bacterial inclusion bodies.
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.
Futami
J
J
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Okayama 700-8530, Japan.
Tsushima
Y
Y
Tada
H
H
Seno
M
M
Yamada
H
H
D001426:Bacterial Proteins / Q000737:chemistry*; D002412:Cations / Q000378:metabolism; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000737:chemistry; D005346:Fibroblast Growth Factors / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D012965:Sodium Chloride / Q000378:metabolism
D001426:Bacterial Proteins; D002412:Cations; D004220:Disulfides; D011994:Recombinant Proteins; D012965:Sodium Chloride; D005346:Fibroblast Growth Factors; D012260:Ribonucleases; D005990:Glycerol