Overexpression, purification, and crystallization of the membrane-bound fumarate reductase from Escherichia coli.
Quinol-fumarate reductase (QFR) from Escherichia coli is a membrane-bound four-subunit respiratory protein that shares many physical and catalytic properties with succinate-quinone oxidoreductase (EC 1.3.99.1) commonly referred to as Complex II. The E. coli QFR has been overexpressed using plasmid vectors so that more than 50% of the cytoplasmic membrane fraction is composed of the four-subunit enzyme complex. The growth characteristics required for optimal levels of expression with minimal degradation by host cell proteases and oxidation factors were determined for the strains harboring the recombinant plasmid. The enzyme is extracted from the enriched membrane fraction using the nonionic detergent Thesit (polyoxyethylene(9)dodecyl ether) in a monodisperse form and then purified by a combination of anion-exchange, perfusion, and gel filtration chromatography. The purified enzyme is highly active and contains all types of redox cofactors expected to be associated with the enzyme. Crystallization screening of the purified QFR by vapor diffusion resulted in the formation of crystals within 24 h using a sodium citrate buffer and polyethylene glycol precipitant. The crystals contain the complete four-subunit QFR complex, diffract to 3.3 A resolution, and were found to be in space group P2(1)2(1)2(1) with unit cell dimensions a = 96.6 A, b = 138.1 A, and c = 275.3 A. The purification and crystallization procedures are highly reproducible and the general procedure may prove useful for Complex IIs from other sources.
Luna-Chavez
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C
Molecular Biology Division (151-S), VA Medical Center, San Francisco, California 94121, USA.
Iverson
T M
TM
Rees
D C
DC
Cecchini
G
G
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013385:Succinate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014812:Vitamin K / Q000737:chemistry
D001426:Bacterial Proteins; D008565:Membrane Proteins; D014812:Vitamin K; D013385:Succinate Dehydrogenase