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Optimization of Expression and Purification of Recombinant Archeoglobus fulgidus FH: NADP Oxidoreductase, an F Cofactor Dependent Enzyme

Protein Journal. 2015; 
Le, Cuong; Joseph, Ebenezer; Nguyen, Toan; Johnson-Winters, Kayunta
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Custom Vector Construction … sequence of A fulgidus Fno (NCBI Protein Database GI:74513452) was used to derive a DNA coding sequence with codons optimized for heterologous expres- sion in E coli, and this sequence was synthesized and incorporated into a shuttle vector by GenScript USA Inc … Get A Quote

摘要

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), which can be interconverted between its reduced and oxidized forms by the FH: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 puri... More

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