Role of Stimuli-Sensitive Polymers in Protein Refolding: α-Amylase and CcdB (Controller of Cell Division or Death B) as Model Proteins
| Autor: | Bipasha Barua, Munishwar N. Gupta, Smita Raghava, Kalyani Mondal, Raghavan Varadarajan |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
Protein Folding
Cell division Alginates Bacterial Toxins Mutation Missense Inclusion bodies Dithiothreitol chemistry.chemical_compound Bacterial Proteins Glucuronic Acid Polymethacrylic Acids Electrochemistry General Materials Science Methyl methacrylate Spectroscopy Chromatography Aqueous solution biology Chemistry Hexuronic Acids Surfaces and Interfaces Condensed Matter Physics Urea biology.protein Protein folding alpha-Amylases Alpha-amylase |
| Zdroj: | Langmuir. 23:70-75 |
| ISSN: | 1520-5827 0743-7463 |
| DOI: | 10.1021/la0616799 |
| Popis: | Alginate, a calcium-sensitive polymer, could carry out simultaneous purification and refolding of 8 M urea/100 mM dithiothreitol (DTT) denatured and thermally denatured alpha-amylase present in a commercial preparation. Activity recoveries of 80 and 70% in the former and the latter cases, respectively, were obtained. The fluorescence spectra showed refolding, and PAGE showed the absence of any aggregates in the refolded preparation. As another example, Eudragit S-100, a pH-sensitive poly(methyl methacrylate), was used to refold CcdB (controller of cell division or death B) protein. Initial experiments with wild-type (WT) CcdB showed that Eudragit bound and precipitated (upon lowering the pH to 4.0) CcdB quantitatively from the latter's aqueous solution. The bioconjugate showed DNA gyrase inhibition activity of CcdB and could be recycled. The inclusion bodies of CcdB mutant CcdB-17P were solubilized in 8 M urea/100 mM dithiothreitol. This preparation could be refolded by precipitation with Eudragit. The fluorescence and CD spectra showed that protein refolding has occurred. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|