title: Simultaneous refolding and purification of a recombinant lipase with an intein tag by affinity precipitation with chitosan.
abstract: A strategy for simultaneous purification and refolding of proteins overexpressed with an intein tag is described. A recombinant lipase overexpressed in Escherichia coli ER2566 with the intein tag and obtained as inclusion bodies was solubilized in buffer containing 8 M urea or cetyltrimethylammonium bromide. The solubilized lipase was precipitated with chitosan and the affinity complex of the polymer with the fusion protein was obtained. The intein tag was cleaved with dithiothreitol and the refolded lipase was obtained in active form. Activity recovery of 80% was observed and the enzyme had a specific activity of 2965 units/mg. The purified lipase showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purity and activity recovery were comparable with that of the preparation obtained by using the commercial kit which utilizes chromatography on chitin beads. The purified and refolded lipase was characterized by fluorescence and CD spectroscopy.
authors: Chemistry Department, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India.
authors: Pradeep K
authors: PK
authors: Singh
authors: Munishwar N
authors: MN
authors: Gupta
mesh_terms: D016956:Burkholderia cepacia / Q000201:enzymology; D011232:Chemical Precipitation; D002686:Chitin / Q000378:metabolism / Q000494:pharmacology; D048271:Chitosan / Q000494:pharmacology*; D002846:Chromatography, Affinity / Q000379:methods*; D004526:Efficiency; D047668:Inteins / Q000502:physiology; D008049:Lipase / Q000737:chemistry* / Q000302:isolation & purification*; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D011993:Recombinant Fusion Proteins; D002686:Chitin; D048271:Chitosan; D008049:Lipase