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bioRxiv · 10.1101/811059

Elevated Expression of a Functional Suf Pathway in the E. coli BL21(DE3) Cell Line Enhances Recombinant Production of an Iron-Sulfur Cluster Containing Protein.

Corless, E. I.·Mettert, E. L.·Kiley, P. J.·Antony, E.
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Abstract

Structural and spectroscopic analysis of iron-sulfur [Fe-S] cluster-containing proteins is often limited by the occupancy and yield of recombinantly produced proteins. Here we report that Escherichia coli BL21(DE3), a strain routinely used to overexpress [Fe-S] cluster-containing proteins, has a nonfunctional Suf pathway, one of two E. coli [Fe-S] cluster biogenesis pathways. We confirmed that BL21(DE3) and commercially available derivatives carry a deletion that results in an inframe fusion of sufA and sufB genes within the sufABCDSE operon. We show that this fusion protein accumulates in cells but is - inactive in [Fe-S] biogenesis. Restoration of an intact Suf pathway combined with enhanced suf operon expression led to a remarkable (~3-fold) increase in the production of the [4Fe-4S] cluster-containing BchL protein, a key component of the dark-operative protochlorophyllide oxido-reductase complex. These results show that this engineered SufFeScient derivative of BL21(DE3) is suitable for enhanced large-scale synthesis of an [Fe-S] cluster-containing protein.\n\nIMPORTANCELarge quantities of recombinantly overexpressed iron-sulfur cluster-containing proteins are necessary for their in-depth biochemical characterization. Commercially available E. coli strain BL21(DE3) and its derivatives have a mutation that inactivates the function of one of the two native pathways (Suf pathway) responsible for cluster biogenesis. Correction of the mutation, combined with sequence changes that increase Suf expression can increase yield and cluster occupancy of [Fe-S] cluster-containing enzymes, facilitating the biochemical analysis of this fascinating group of proteins.

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Corless, E. I., Mettert, E. L., Kiley, P. J., Antony, E.. 2019-10-18. Elevated Expression of a Functional Suf Pathway in the E. coli BL21(DE3) Cell Line Enhances Recombinant Production of an Iron-Sulfur Cluster Containing Protein.. https://doi.org/10.1101/811059

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