title: An efficient in vitro refolding of recombinant bacterial laccase in Escherichia coli.
abstract: Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are important multicopper enzymes that are used in many biotechnological processes. A recombinant form of laccase from Bacillus sp. HR03 was overexpressed in Escherichia coli BL-21(DE3). Inclusion body (IB) formation happens quite often during recombinant protein production. Hence, developing a protocol for efficient refolding of proteins from inclusion bodies to provide large amounts of active protein could be advantageous for structural and functional studies. Here, we have tried to find an efficient method of refolding for this bacterial enzyme. Solubilization of inclusion bodies was carried out in phosphate buffer pH 7, containing 8M urea and 4mM β-mercaptoethanol and refolding was performed using the dilution method. The effect of different additives was investigated on the refolding procedure of denaturated laccase. Mix buffer (phosphate buffer and citrate buffer, 100mM) containing 4mM ZnSO4 and 100mM sorbitol was selected as an optimized refolding buffer. Also Kinetic parameters of soluble and refolded laccase were analyzed.
authors.affiliation: Department of Biology, Faculty of Basic Sciences, Hakim Sabzevari University, Sabzevar, Iran.
authors.forename: Nasrin
authors.initials: N
authors.lastname: Mollania
authors.forename: Khosro
authors.initials: K
authors.lastname: Khajeh
authors.forename: Bijan
authors.initials: B
authors.lastname: Ranjbar
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Rashno
authors.forename: Neda
authors.initials: N
authors.lastname: Akbari
authors.forename: Mehrnoosh
authors.initials: M
authors.lastname: Fathi-Roudsari
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001709:Biotechnology / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D042845:Laccase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D013050:Spectrometry, Fluorescence
chemical_list: D011994:Recombinant Proteins; D042845:Laccase