title: High-level soluble expression of a thermostable xylanase from thermophilic fungus Thermomyces lanuginosus in Escherichia coli via fusion with OsmY protein.
abstract: A thermostable xylanase is encoded by xynA from fungus Thermomyces lanuginosus. The problem emerged from overexpression of xynA in Escherichia coli has been the formation of inclusion bodies. Here we describe the xynA was fused with the hyperosmotically inducible periplasmic protein of E. coli, OsmY. The fusion protein OsmY-xynA was expressed as almost all soluble form. The soluble expression level of fusion protein reached 98±6U/ml when cells containing pET-OsmY-xynA were expressed without IPTG induction at 37°C. The induction is probably due to auto-induction due to lactose in the medium (Studier (2005) [21]). The cells harboring pET-OsmY-xynA expressed an activity level about 24 times higher than that expressed from pET-20b-xynA. Xylanase activity was observed in the extracellular (36±1.3U/ml) and the periplasmic (42±4U/ml) when cells containing pET-OsmY-xynA were induced without IPTG addition. After the cold osmotic shock procedure followed by nickel affinity chromatography, the purified fusion protein showed a single band on SDS-PAGE gel with a molecular mass of 44kDa. The purified fusion enzyme exhibited the highest activity at 65°C and pH 6.0.
authors.affiliation: School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, PR China. Electronic address: leyilin@163.com.
authors.forename: Yilin
authors.initials: Y
authors.lastname: Le
authors.affiliation: School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, PR China.
authors.forename: Huilei
authors.initials: H
authors.lastname: Wang
mesh_terms: D001203:Ascomycota / Q000201:enzymology; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D005822:Genetic Vectors; D006358:Hot Temperature; D033902:Periplasmic Binding Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
chemical_list: D029968:Escherichia coli Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; C074783:osmY protein, E coli; D043364:Endo-1,4-beta Xylanases
keywords: Escherichia coli; Fusion protein; Soluble expression; Thermomyces lanuginosus; Xylanase