Cloning, expression and purification of copper nitrite reductase

In this study, a novel copper nitrite reductase gene was cloned from Achromobacter xylosoxidans DL-1 by using a PCR protocol.The gene contained a 1083 bp open reading frame encoding a 360 amino acid protein with an estimated molecular mass of 38.924 k Da and isoelectronic point ( p I) of 4.83, named Cu NiR ( Genebank no.KX674378) .Based on domains analysis, Cu NiR had a signal peptide, a copper binding site classified as Cu-oxidase-3, and one nitrite reductase catalytic domain ( Cuoxidase) . A recombinant plasmid, p ET22b-Cu NiR was constructed and transformed into E.coli BL21 ( DE3) . The cells were induced by addition of IPTG. Cu NiR had been successfully expressed by the analysis of sodium dodecyl sulphate-polyacrylamide gel electrophoresis ( SDS-PAGE) . The recombinant protein was purified by Ni-NTA resin, and the specific activity was 123.82 U/mg, which lay the theoretical basis for biochemical characterization of the copper nitrite reductase.