CLONING, SEQUENCING, AND EXPRESSION OF THE GENES ENCODING SUBUNITS OFPARACOCCUS-DENITRIFICANS ELECTRON-TRANSFER FLAVOPROTEIN

The genes encoding the two subunits of Paracoccus denitrificans electr on transfer flavoprotein (ETF) were identified by screening a genomic library constructed in pBluescript II SK+ with probes generated by amp lification of genomic sequences by the polymerase chain reaction. Prim ers for the polymerase chain reaction were designed based on peptide s equences from purified Paracoccus ETF subunits. The genes are arranged in tandem in the genomic DNA with the deoxyadenylic acid residue in t he TGA termination codon of the small subunit providing the deoxyadeny lic acid residue for the ATG initiating codon of the large subunit. Th e deduced amino acid sequences of the ETF subunits exhibits extensive sequence identity with the human ETF subunits. The Paracoccus ETF is e xpressed from the pBluescript vector in Escherichia coli, yielding 30 mg of purified, catalytically active protein per liter of culture.