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Optimization of Expression and Purification of Recombinant Archeoglobus fulgidus F420H2:NADP+ Oxidoreductase, an F420 Cofactor Dependent Enzyme

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Abstract

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.

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Abbreviations

Fno:

F420H2:NADP+ oxidoreductase

NADP+ :

Nicotinamide adenine dinucleotide phosphate

E. coli :

Escherichia coli

Archeoglobus fulgidus :

A. fulgidus

IPTG:

Isopropyl β-D-1-thiogalactopyranoside

FO:

F420 cofactor precursor

LB:

Luria Bertani broth

Tris:

Tris(hydroxymethyl)aminomethane

MES:

2-(N-morpholino)ethanesulfonic acid

SDS–PAGE:

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis

k cat :

Michaelis–Menten catalytic rate constant (turnover number)

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Acknowledgments

This work was supported by the National Science Foundation, Grant number, 1120837 (to KJW). We also thank Drs. John H. Enemark, Graham R. Moran, Frank W. Foss, Richard Timmons and Walter Fast for helpful discussions. Figure 4 (right figure, steady-state kinetics plot with FO) was reproduced from the following reference. Hossain, M. S.; Le, C. Q.; Joseph, E.; Nguyen, T. Q.; Johnson-Winters, K.; Foss, F. W., Jr. Convenient synthesis of deazaflavin cofactor FO and its activity in F420-dependent NADP reductase. Org. Biomol. Chem, 13, 5082–5085. Reproduced by permission of The Royal Society of Chemistry.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX, 76019-0065, USA

    Cuong Quang Le, Ebenezer Joseph, Toan Nguyen & Kayunta Johnson-Winters

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  1. Cuong Quang Le
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  2. Ebenezer Joseph
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  3. Toan Nguyen
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  4. Kayunta Johnson-Winters
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Correspondence to Kayunta Johnson-Winters.

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Le, C.Q., Joseph, E., Nguyen, T. et al. Optimization of Expression and Purification of Recombinant Archeoglobus fulgidus F420H2:NADP+ Oxidoreductase, an F420 Cofactor Dependent Enzyme. Protein J 34, 391–397 (2015). https://doi.org/10.1007/s10930-015-9633-y

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