Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein

Corless, Elliot I.; Mettert, Erin L.; Kiley, Patricia J.; Antony, Edwin; Metcalf, William W.

doi:10.1128/JB.00496-19

Title: Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein

Journal Article · 10 November 2019 · Journal of Bacteriology

DOI: https://doi.org/10.1128/JB.00496-19 · OSTI ID:1593945

Corless, Elliot I. ^[1]; Mettert, Erin L. ^[2]; Kiley, Patricia J. ^[2]; Antony, Edwin ^[3]; Metcalf, William W. ^[4]

Marquette Univ., Milwaukee, WI (United States); Saint Louis University
Univ. of Wisconsin, Madison, WI (United States)
Marquette Univ., Milwaukee, WI (United States); Saint Louis Univ. School of Medicine, MO (United States)
Univ. of Illinois at Urbana-Champaign, IL (United States)

Structural and spectroscopic analysis of iron-sulfur [Fe-S] cluster-containing proteins is often limited by the occupancy and yield of recombinantly produced proteins. In this work, we report that Escherichia coli BL21(DE3), a strain routinely used to overproduce [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 in-frame 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] cluster 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 oxidoreductase 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.

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Research Organization:: Marquette Univ., Milwaukee, WI (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)

Grant/Contract Number:: SC0017866

OSTI ID:: 1593945

Journal Information:: Journal of Bacteriology, Journal Name: Journal of Bacteriology Journal Issue: 3 Vol. 202; ISSN 0021-9193

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
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Title: Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein

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