Thioredoxin reductase from Escherichia coli: Evidence of restriction to a single conformation upon formation of a crosslink between engineered cysteines

ABSTRACT Release factor 2 (RF2), encoded by the prfB gene in Escherichia coli, catalyzes translational termination at UGA and UAA codons. Termination at UGA competes with selenocysteine (Sec) incorporation at Sec-dedicated UGA codons, and RF2 thereby counteracts expression of selenoproteins. prfB is an essential gene in E. coli and can therefore not be removed in order to increase yield of recombinant selenoproteins. We therefore constructed an E. coli strain with the endogenous chromosomal promoter of prfB replaced with the titratable PBAD promoter. Knockdown of prfB expression gave a bacteriostatic effect, while two- to sevenfold overexpression of RF2 resulted in a slightly lowered growth rate in late exponential phase. In a turbidostatic fermentor system the simultaneous impact of prfB knockdown on growth and recombinant selenoprotein expression was subsequently studied, using production of mammalian thioredoxin reductase as model system. This showed that lowering the levels of RF2 correlated directly with increasing Sec incorporation specificity, while also affecting total selenoprotein yield concomitant with a lower growth rate. This study thus demonstrates that expression of prfB can be titrated through targeted exchange of the native promoter with a PBAD-promoter and that knockdown of RF2 can result in almost full efficiency of Sec incorporation at the cost of lower total selenoprotein yield.

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Crystal Structure of Escherichia coli Thioredoxin Reductase Refined at 2 Å Resolution

Journal of Molecular Biology ◽

10.1006/jmbi.1994.1190 ◽

1994 ◽

Vol 236 (3) ◽

pp. 800-816 ◽

Cited By ~ 134

Author(s):

Gabriel Waksman ◽

Talluru S.R. Krishna ◽

Charles H. Williams ◽

John Kuriyan

Keyword(s):

Crystal Structure ◽

Escherichia Coli ◽

Thioredoxin Reductase

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Mechanism and structure of thioredoxin reductase from Escherichia coli

The FASEB Journal ◽

10.1096/fasebj.9.13.7557016 ◽

1995 ◽

Vol 9 (13) ◽

pp. 1267-1276 ◽

Cited By ~ 105

Author(s):

Charles H. Williams

Keyword(s):

Escherichia Coli ◽

Thioredoxin Reductase

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Bacterial and mammalian thioredoxin systems activate iodothyronine 5′-deiodination

Biochemistry and Cell Biology ◽

10.1139/o88-057 ◽

1988 ◽

Vol 66 (5) ◽

pp. 460-464 ◽

Cited By ~ 7

Author(s):

Arun K. Das ◽

Brian C. W. Hummel ◽

Florence K. Gleason ◽

Arne Holmgren ◽

Paul G. Walfish

Keyword(s):

Escherichia Coli ◽

Rat Liver ◽

Liver Microsomes ◽

Thioredoxin Reductase ◽

Relative Mass ◽

Rat Liver Microsomes ◽

Liver Cytosol ◽

E Coli ◽

Thioredoxin System ◽

Rat Liver Cytosol

The identity of a dithiol (designated DFB) of relative mass (Mr) = 13 000, reported previously to be present infraction B of rat liver cytosol and to participate as a cofactor in the 5′-deiodination of iodothyronines, has been investigated. Substitution of highly purified thioredoxin from Escherichia coli for fraction B or of highly purified thioredoxin reductase from either E. coli or rat liver for cytosolic fraction A (containing DFB reductase) permits deiodination of 3,3′,5′-[l25I]triiodothyronine by rat liver microsomes to proceed. Addition of antibodies to highly purified rat-liver thioredoxin or thioredoxin reductase inhibits deiodination. Thus, the thioredoxin system largely accounts for the activity of the cytosolic cofactor system supporting 5′-deiodination of 3,3′,5′-triiodothyronine in rat liver.

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