Simultaneous expression and maturation of the iron‐sulfur protein ferredoxin in a cell‐free system

Abstract

The model iron-sulfur (Fe-S) protein ferredoxin (Fd) from Synechocystis sp. PCC 6803 has been simultaneously produced and matured in a cell-free production system. After 6 h of incubation at 37 degrees C, Fd accumulated to >450 microg/mL. Essentially all was soluble, and 85% was active. Production and maturation of the protein in the cell-free system were found to be dependent in a coupled manner on the concentration of the supplemented iron and sulfur sources, ferrous ammonium sulfate and cysteine, respectively. The recombinant expression of ISC helper proteins during cell extract preparation did not increase cell-free Fd accumulation or activity, although the efficiency of iron and cysteine utilization increased. Fd maturation was independent of protein production rate, and proceeded at a constant rate throughout the period of active translation. In addition, incubation of denatured apo Fd with cell-free reaction components resulted in recovery of Fd activity, supporting the interpretation that maturation mechanisms did not act co-translationally. Incubation at 28 degrees C increased total and active protein accumulation, but decreased the ratio of active to total Fd produced. In summary, the high product yields and folding efficiency make the cell-free system described here an attractive platform for the study of Fe-S protein production and maturation. The system enables both small-volume, high throughput investigations as well as larger scale production. To our knowledge, this is the first demonstration of directed, high-yield production and maturation of an Fe-S protein in a cell-free system.

High-Yield Expression of Heterologous [FeFe] Hydrogenases in Escherichia coli

“…The petF gene from Synechocystis sp. PCC 6803 was PCR amplified from the pK7 expression vector and cloned into the pET-21(b) plasmid [41]. All expression constructs were confirmed by DNA sequencing and transformed into the E. coli strains BL21(DE3) (Novagen) and BL21(DE3) ΔiscR by selection with the appropriate antibiotics.…”

“…PCC 6803 was first cloned from the pK7 plasmid [40] into the pET-21(b) vector, and subsequently transformed into BL21(DE3) ΔiscR . Both PetF expression and purification using ammonium sulfate precipitation followed by anion exchange chromatography were carried out as previously described [41], [44].…”

Cell‐free synthesis and maturation of [FeFe] hydrogenases

“…We expect that similar modifications to cell-free systems will have direct applications for the study of other metalloproteins, oxygen-sensitive proteins, and proteins requiring helpers to mature. Additionally, the cell-free system has proven to be adaptable for the production of mammalian proteins (Yang et al, 2005), proteins requiring disulfide bonds (Yin and Swartz, 2004), proteins requiring multiple subunits, proteins requiring [Fe-S] centers and other cofactors (Boyer et al, 2006;Knapp and Swartz, 2004), membrane proteins, proteome libraries, and proteins requiring chaperones (Yin and Swartz, 2004). In combination, these capabilities establish this system as a highly adaptable platform for the production and study of many complex proteins.…”

“…Cell extracts have been improved through strain engineering and modified preparation procedures to enhance the cellfree production of many types of proteins (Boyer et al, 2006;Knapp and Swartz, 2004;Miyazaki-Imamura et al, 2003;Yang et al, 2004;Yin and Swartz, 2004). Two unprecedented adaptations in extract preparation were implemented for expression of active hydrogenase proteins: the inclusion of heterologous maturation enzymes, and the preparation of extracts under strictly anaerobic conditions.…”

“…This allows facile addition of substrates, even to concentrations unachievable in vivo. The ability to control the expression environment has enabled production of proteins requiring [Fe-S] clusters (Boyer et al, 2006), FAD cofactors (Knapp and Swartz, 2004), heme groups (Miyazaki-Imamura et al, 2003), and disulfide bonds (Yin and Swartz, 2004). Since modern cell-free systems more closely mimic the natural cytoplasmic folding environment of cells (Jewett and Swartz, 2004), protein folding can occur co-translationally, and activation may be assisted by intact pathways from E. coli.…”