Role of Stimuli-Sensitive Polymers in Protein Refolding: \alpha-Amylase and CcdB (Controller of Cell Division or Death B) as Model Proteins

Mondal, Kalyani and Raghava, Smita and Barua, Bipasha and Varadarajan, Raghavan and Gupta, Munishwar N (2007) Role of Stimuli-Sensitive Polymers in Protein Refolding: \alpha-Amylase and CcdB (Controller of Cell Division or Death B) as Model Proteins. In: Langmuir, 23 (1). pp. 70-75.

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Abstract

Alginate, a calcium-sensitive polymer, could carry out simultaneous purification and refolding of 8 M urea/100 mM dithiothreitol (DTT) denatured and thermally denatured R-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.

Item Type:	Journal Article
Publication:	Langmuir
Publisher:	American Chemical Society
Additional Information:	Copyright of this article belongs to the American Chemical Society
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	12 Mar 2007
Last Modified:	19 Sep 2010 04:34
URI:	http://eprints.iisc.ac.in/id/eprint/9684

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