title: Titration and conditional knockdown of the prfB gene in Escherichia coli: effects on growth and overproduction of the recombinant mammalian selenoprotein thioredoxin reductase.
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 P(BAD) 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 P(BAD)-promoter and that knockdown of RF2 can result in almost full efficiency of Sec incorporation at the cost of lower total selenoprotein yield.
authors.affiliation: Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
authors.forename: Olle
authors.initials: O
authors.lastname: Rengby
authors.forename: Elias S J
authors.initials: ES
authors.lastname: Arnér
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003062:Codon; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development*; D029968:Escherichia coli Proteins / Q000235:genetics*; D017353:Gene Deletion; D008969:Molecular Sequence Data; D010454:Peptide Termination Factors / Q000235:genetics*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis; D017279:Selenocysteine / Q000378:metabolism; D051140:Selenoproteins / Q000235:genetics / Q000378:metabolism*; D013880:Thioredoxin-Disulfide Reductase / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D003062:Codon; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; D011994:Recombinant Proteins; D051140:Selenoproteins; C489006:prfB protein, E coli; D017279:Selenocysteine; D013880:Thioredoxin-Disulfide Reductase