Cloning, sequencing, and expression of the genes encoding subunits of Paracoccus denitrificans electron transfer flavoprotein.

Abstract

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