OVEREXPRESSION IN ESCHERICHIA-COLI OF A RECOMBINANT CHIMERIC RHODOTORULA-GRACILIS D-AMINO-ACID OXIDASE

This paper reports a novel expression system constructed to maximize t he production in Escherichia coli of D-amino acid oxidase from the yea st Rhodotorula gracilis (RgDAAO). We produced a recombinant plasmid by the insertion of the cDNA encoding for the RgDAAO into the multiple c loning site of the expression vector pT7.7 (pT7-DAAO), downstream of t he 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. col i cells. In the latter host and under optimal IPTG induction condition s, 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 fermenta tion broth), with a specific activity of 8.8 U/mg protein. RgDAAO repr esents approximate to 8% of the total soluble protein content of the c ell. (C) 1998 Academic Press.