Nov, 2000
Important parameters affecting efficiency of protein refolding by reversed micelles
BIOTECHNOLOGY PROGRESS
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- Volume
- 16
- Number
- 6
- First page
- 1079
- Last page
- 1085
- Language
- English
- Publishing type
- DOI
- 10.1021/bp000073m
- Publisher
- AMER CHEMICAL SOC
Refolding of denatured RNase A as a model of inclusion bodies was performed by reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) in isooctane. In the novel refolding process, a solid-liquid extraction was utilized as an alternative to the ordinary protein extraction by reversed micelles based on a liquid-liquid extraction. First, the effects of operational parameters such as concentration of AOT, W-o (= [H2O]/[AOT]), and pH were examined on the solubilization of solid denatured proteins into a reversed micellar solution. The solubilization was facilitated by a high AOT concentration, a high W-o value, and a high pH in water pools. These conditions are favorable for the dispersion of the solid protein aggregates in an organic solvent. Second, the renaturation of the denatured RNase A solubilized into the reversed micellar solution was conducted by addition of glutathione as a redox reagent. A complete renaturation of RNase A was accomplished by adjusting the composition of the redox reagent even at a high protein concentration in which protein aggregation would usually occur in aqueous media. In addition, the renaturation rates were improved by optimizing water content (W-o) and the pH of water pools in reversed micelles. Finally, the recovery of renatured RNase A from the reversed micellar solution was performed by adding a polar organic solvent such as acetone into the reversed micellar solution. This precipitation method was effective for recovering proteins from reversed micellar media without any significant reduction in enzymatic activity.
- Link information
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- DOI
- https://doi.org/10.1021/bp000073m
- CiNii Articles
- http://ci.nii.ac.jp/naid/30011446017
- PubMed
- https://www.ncbi.nlm.nih.gov/pubmed/11101337
- Web of Science
- https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000165719000022&DestApp=WOS_CPL
- ID information
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- DOI : 10.1021/bp000073m
- ISSN : 8756-7938
- CiNii Articles ID : 30011446017
- Pubmed ID : 11101337
- Web of Science ID : WOS:000165719000022