title: Heterologous metalloprotein biosynthesis in Escherichia coli: conditions for the overproduction of functional copper-containing nitrite reductase and azurin from Alcaligenes xylosoxidans.
abstract: This paper reports on the optimization of conditions for the overproduction and isolation of two recombinant copper metalloproteins, originally encoded on the chromosome of the dentrifying soil bacterium Alcaligenes xylosoxidans, in the heterologous host Escherichia coli. The trimeric enzyme nitrite reductase (NiR) contains both type-1 and type-2 Cu centres, whilst its putative redox partner, azurin I, is monomeric and has only a type-1 Cu centre. Both proteins were processed and exported to the periplasm of E. coli, which is consistent with their periplasmic location in their native host A. xylosoxidans. NiR could be readily purified from the periplasmic fraction of E. coli but the enzyme as isolated possessed only type-1 Cu centres. The type-2 Cu centre could be fully reconstituted by incubation of the periplasmic fraction with copper sulfate prior to enzyme purification. Azurin I could only be isolated with a fully occupied type-1 centre when isolated from the crude cell extract but not after isolation from the periplasmic fraction, suggesting loss of the copper due to proteolysis. Based on a number of criteria, including spectroscopic, mass spectrometric, biochemical and structural analyses, both recombinant proteins were found to be indistinguishable from their native counterparts isolated from A. xylosoxidans. The findings of this work have important implications for the overproduction of recombinant metalloproteins in heterologous hosts.
authors.affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK.
authors.forename: Roger L
authors.initials: RL
authors.lastname: Harris
authors.forename: Miguel
authors.initials: M
authors.lastname: Prudêncio
authors.forename: S Samar
authors.initials: SS
authors.lastname: Hasnain
authors.forename: Robert R
authors.initials: RR
authors.lastname: Eady
authors.forename: R Gary
authors.initials: RG
authors.lastname: Sawers
mesh_terms: D000421:Alcaligenes / Q000201:enzymology / Q000235:genetics*; D001400:Azurin / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D003300:Copper; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D005798:Genes, Bacterial; D008667:Metalloproteins / Q000096:biosynthesis* / Q000737:chemistry; D009565:Nitrate Reductases / Q000096:biosynthesis / Q000235:genetics*; D019897:Periplasm; D011487:Protein Conformation
chemical_list: D008667:Metalloproteins; D001400:Azurin; D003300:Copper; D009565:Nitrate Reductases