title: Overexpression in Escherichia coli of a recombinant chimeric Rhodotorula gracilis d-amino acid oxidase.
abstract: This paper reports a novel expression system constructed to maximize the production in Escherichia coli of d-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO). We produced a recombinant plasmid by the insertion of the cDNA encoding for the RgDAAO into the multiple cloning site of the expression vector pT7.7 (pT7-DAAO), downstream of the T7 RNA polymerase binding site. The pT7-DAAO, which encodes a fully active fusion protein with six additional residues at the N-terminus of DAAO, was used to transform the BL21(DE3) and BL21(DE3)pLysS E. coli cells. In the latter host and under optimal IPTG induction conditions, soluble and active chimeric DAAO was expressed in these cells up to 930 U/g of cell (and a fermentation yield of 2300 U/liter of fermentation broth), with a specific activity of 8.8 U/mg protein. RgDAAO represents approximately 8% of the total soluble protein content of the cell.
authors.affiliation: Department of Structural and Functional Biology, University of Insubria, via J. H. Dunant 3, Varese, 21100, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Molla
authors.forename: C
authors.initials: C
authors.lastname: Vegezzi
authors.forename: M S
authors.initials: MS
authors.lastname: Pilone
authors.forename: L
authors.initials: L
authors.lastname: Pollegioni
mesh_terms: D000409:Alanine / Q000378:metabolism; D003605:D-Amino-Acid Oxidase / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry; D015870:Gene Expression / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012248:Rhodotorula / Q000201:enzymology*
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D003605:D-Amino-Acid Oxidase; D000409:Alanine