Optimization of Expression and Purification of Recombinant Archeoglobus fulgidus F.sub.420H.sub.2:NADP.sup.+ Oxidoreductase, an F.sub.420 Cofactor Dependent Enzyme

Methanogens play a critical role in carbon cycling and contain a number of intriguing biosynthetic pathways. One unusual cofactor found in methanogenic and sulfate reducing archaea is Factor 420 (F.sub.420), which can be interconverted between its reduced and oxidized forms by the F.sub.420H.sub.2:NADP.sup.+ oxidoreductase (Fno) through hydride transfer mechanisms. Here, we report an optimized expression and purification method for recombinant Fno derived from the extreme thermophile Archeoglobus fulgidus. An expression vector that is codon-optimized for heterologous expression in Escherichia coli, modified growth conditions, and a modified purification protocol involving a key polyethyleneimine precipitation step results in a highly purified, homogeneous preparation of Fno that displays high catalytic activity with a truncated F.sub.420 analog. This method should accelerate studies on how Fno uses the unusual F.sub.420 cofactor during catalysis.