Molla G, Vegezzi C, Pilone M S, Pollegioni L
Department of Structural and Functional Biology, University of Insubria, via J. H. Dunant 3, Varese, 21100, Italy.
Protein Expr Purif. 1998 Nov;14(2):289-94. doi: 10.1006/prep.1998.0956.
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.
本文报道了一种新型表达系统，该系统构建的目的是使来自纤细红酵母（RgDAAO）的d-氨基酸氧化酶在大肠杆菌中的产量最大化。我们通过将编码RgDAAO的cDNA插入表达载体pT7.7的多克隆位点（pT7-DAAO），在T7 RNA聚合酶结合位点的下游，构建了一个重组质粒。pT7-DAAO编码一种在DAAO的N端带有六个额外残基的完全活性融合蛋白，用于转化BL21(DE3)和BL21(DE3)pLysS大肠杆菌细胞。在后者宿主及最佳IPTG诱导条件下，可溶性且有活性的嵌合DAAO在这些细胞中表达，产量高达930 U/g细胞（发酵产率为2300 U/升发酵液），比活性为8.8 U/mg蛋白。RgDAAO约占细胞总可溶性蛋白含量的8%。
