Optimization of Expression and Purification of Recombinant Archeoglobus fulgidus F420H2:NADP+ Oxidoreductase, an F420 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 (F420), which can be interconverted between its reduced and oxidized forms by the F420H2:NADP(+) 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 F420 analog. This method should accelerate studies on how Fno uses the unusual F420 cofactor during catalysis.
Cuong Quang
CQ
Le
Ebenezer
E
Joseph
Toan
T
Nguyen
Kayunta
K
Johnson-Winters
D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D019609:Archaeoglobus / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; C109941:F420-dependent NADP reductase