Overexpression, Purification, and Crystallization of the Membrane-Bound Fumarate Reductase from Escherichia coli

2000 ◽  
Vol 19 (1) ◽  
pp. 188-196 ◽  
Author(s):  
César Luna-Chavez ◽  
Tina M. Iverson ◽  
Douglas C. Rees ◽  
Gary Cecchini
Keyword(s):  
Escherichia Coli ◽  
Fumarate Reductase ◽  
Membrane Bound

scholarly journals The effects of anions on fumarate reductase isolated from the cytoplasmic membrane of Escherichia coli

1981 ◽  
Vol 199 (3) ◽  
pp. 473-477 ◽  
Author(s):  
J J Robinson ◽  
J H Weiner
Keyword(s):  
Escherichia Coli ◽  
Cytoplasmic Membrane ◽  
Chemical Properties ◽  
Fumarate Reductase ◽  
Maximal Velocity ◽  
Nitrobenzoic Acid ◽  
Thiol Reagent ◽  
Membrane Bound ◽  
Physical And Chemical ◽  
And Chemical Properties

A broad range of anions was shown to stimulate the maximal velocity of purified fumarate reductase isolated from the cytoplasmic membrane of Escherichia coli, while leaving the Km for fumarate unaffected. Reducing agents potentiate the effects of anions on the activity, but have no effect by themselves. Thermal stability, conformation as monitored by circular dichroism and susceptibility to the thiol reagent 5,5′-dithiobis-(2-nitrobenzoic acid) are also altered by anions. The apparent Km for succinate in the reverse reaction (succinate dehydrogenase activity) varies as a function of anion concentration, but the maximal velocity is not affected. The membrane-bound activity is not stimulated by anions and its properties closely resemble those of the purified enzyme in the presence of anions. Thus it appears that anions alter the physical and chemical properties of fumarate reductase, so that it more closely resembles the membrane-bound form.

Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli

1979 ◽  
Vol 57 (6) ◽  
pp. 813-821 ◽  
Author(s):  
Peter Dickie ◽  
Joel H. Weiner
Keyword(s):  
Escherichia Coli ◽  
Reductase Activity ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sodium Cholate ◽  
Defined Medium ◽  
Exchange Chromatography ◽  
Fumarate Reductase ◽  
Molar Ratios ◽  
Membrane Bound

Fumarate reductase has been purified 100-fold to 95% homogeneity from the cytoplasmic membrane of Escherichia coli, grown anaerobically on a defined medium containing glycerol plus fumarate. Optimal solubilization of total membrane protein and fumarate reductase activity occurred with nonionic detergents having a hydrophobic–lipophilic balance (HLB) number near 13 and we routinely solubilized the enzyme with Triton X-100 (HLB number = 13.5). Membrane enzyme extracts were fractionated by hydrophobic-exchange chromatography on phenyl Sepharose CL-4B to yield purified enzyme. The enzyme, whether membrane bound, in Triton extracts, or purified, had an apparent Km near 0.42 mM. Two peptides with molecular weights of 70 000 and 24 000, present in 1:1 molar ratios, were identified by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis to coincide with enzyme activity. A minimal native molecular weight of 100 000 was calculated for fumarate reductase by Sephacryl S-200 gel filtration in the presence of sodium cholate. This would indicate that the enzyme is a dimer. The purified enzyme has low, but measurable, succinate dehydrogenase activity.

scholarly journals Identification of membrane anchor polypeptides of Escherichia coli fumarate reductase

1982 ◽  
Vol 152 (3) ◽  
pp. 1126-1131
Author(s):  
B D Lemire ◽  
J J Robinson ◽  
J H Weiner
Keyword(s):  
Escherichia Coli ◽  
Gene Cloning ◽  
Nonheme Iron ◽  
Fumarate Reductase ◽  
Alkaline Ph ◽  
Membrane Anchor ◽  
E Coli ◽  
Molecular Weights ◽  
Membrane Bound ◽  
Triton X

Fumarate reductase of Escherichia coli has been shown to be a membrane-bound enzyme composed of a 69,000-dalton catalytic-flavin-containing subunit and a 27,000-dalton nonheme-iron-containing subunit. Using gene cloning and amplification techniques, we have observed two additional polypeptides encoded by the frd operon, with apparent molecular weights of 15,000 and 14,000, which are expressed when E. coli is grown anaerobically on glycerol plus fumarate. Expression of these two small polypeptides is necessary for the two large subunits to associate with the membrane. The four subunits remain associated in Triton X-100 extracts of the membrane, and a holoenzyme form of fumarate reductase containing one copy of each of the four polypeptides has been isolated. Unlike the well-characterized two-subunit form, the holoenzyme is not dependent on anions for activity and is not labile at alkaline pH. In these respects, it more closely resembles the membrane-bound activity.

Conformational properties of membrane-bound fumarate reductase of Escherichia coli

1986 ◽  
Vol 249 (2) ◽  
pp. 579-587 ◽  
Author(s):  
Clara Fronticelli ◽  
Enrico Bucci ◽  
Arthur Zachary ◽  
Barry P. Rosen
Keyword(s):  
Escherichia Coli ◽  
Fumarate Reductase ◽  
Conformational Properties ◽  
Membrane Bound

scholarly journals Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jasmine M. Hershewe ◽  
Katherine F. Warfel ◽  
Shaelyn M. Iyer ◽  
Justin A. Peruzzi ◽  
Claretta J. Sullivan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Synthetic Biology ◽  
Membrane Protein ◽  
Membrane Vesicles ◽  
Processing Methods ◽  
Glycoprotein Synthesis ◽  
On Demand ◽  
Free Gene ◽  
Membrane Bound

AbstractCell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

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