Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein
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. 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.
- Authors:
-
- Marquette Univ., Milwaukee, WI (United States)
- 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)
- Publication Date:
- Research Org.:
- Marquette Univ., Milwaukee, WI (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Basic Energy Sciences (BES)
- OSTI Identifier:
- 1593945
- Alternate Identifier(s):
- OSTI ID: 1827181
- Grant/Contract Number:
- SC0017866; R01-GM115894
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Journal of Bacteriology
- Additional Journal Information:
- Journal Volume: 202; Journal Issue: 3; Journal ID: ISSN 0021-9193
- Publisher:
- American Society for Microbiology
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; electron transfer; Fe-S protein overproduction; E. coli B; iron-sulfur biogenesis; Suf pathway; Fe-S cluster biogenesis; Fe-S protein overexpression; Suf; Photosynthesis; DPOR; Nitrogenase; Nitrogenase-like enzymes; FeS
Citation Formats
Corless, Elliot I., Mettert, Erin L., Kiley, Patricia J., Antony, Edwin, and Metcalf, William W. Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein. United States: N. p., 2019.
Web. doi:10.1128/JB.00496-19.
Corless, Elliot I., Mettert, Erin L., Kiley, Patricia J., Antony, Edwin, & Metcalf, William W. Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein. United States. https://doi.org/10.1128/JB.00496-19
Corless, Elliot I., Mettert, Erin L., Kiley, Patricia J., Antony, Edwin, and Metcalf, William W. Mon .
"Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein". United States. https://doi.org/10.1128/JB.00496-19. https://www.osti.gov/servlets/purl/1593945.
@article{osti_1593945,
title = {Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein},
author = {Corless, Elliot I. and Mettert, Erin L. and Kiley, Patricia J. and Antony, Edwin and Metcalf, William W.},
abstractNote = {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.},
doi = {10.1128/JB.00496-19},
journal = {Journal of Bacteriology},
number = 3,
volume = 202,
place = {United States},
year = {2019},
month = {11}
}
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