Role of stimuli-sensitive polymers in protein refolding: alpha-amylase and CcdB (controller of cell division or death B) as model proteins.
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.
Mondal
Kalyani
K
Chemistry Department, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India.
Raghava
Smita
S
Barua
Bipasha
B
Varadarajan
Raghavan
R
Gupta
Munishwar N
MN
D000464:Alginates / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D020723:Glucuronic Acid / Q000737:chemistry; D006603:Hexuronic Acids / Q000737:chemistry; D020125:Mutation, Missense; D011109:Polymethacrylic Acids / Q000737:chemistry*; D017510:Protein Folding; D000516:alpha-Amylases / Q000737:chemistry*
D000464:Alginates; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068356:CcdB protein, Plasmid F; D006603:Hexuronic Acids; D011109:Polymethacrylic Acids; C038300:methylmethacrylate-methacrylic acid copolymer; D020723:Glucuronic Acid; D000516:alpha-Amylases