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.Studies involving titration of expression of essential Escherichia coli genes must be carefully conducted and naturally cannot be performed using a direct gene knockout procedure. It was shown early that the P BAD promoter is suitable for titration or conditional knockdown of essential genes when used in plasmid-driven complementation of chromosomal gene deletions (13). Recently, additional approaches for chromosomal integration of P BAD aiming at titration and knockdown of essential genes have been described (25,26). One caveat in low-level titration of the transcriptional activity of P BAD (in contrast to knockdown with glucose) is that it generally occurs by an on-or-off effect in individual cells, due to arabinoseinduced induction of the araE transporter, which is needed for uptake of arabinose as an inducer of the P BAD promoter (28). This effect, eliminating the possibility of controlled expressional titration on the cellular level, can be circumvented by use of the BW27783 strain, which has araE constitutively expressed (20). We therefore selected the BW27783 strain as an E. coli host in this work, where we wished to study the impact of the essential (15, 18) prfB gene on selenoprotein expression, utilizing replacement of its native chromosomal promoter with P BAD .Protein translation occurs at the ribosome up to the point of termination of translation, which requires release factor 2 (RF2) as one of two polypeptide chain release factors. Thus, when the protein translation complex reaches an in-frame termination codon in the mRNA and this is exposed at the A site, binding of either RF1, at UAG or UAA codons, or RF2, a...

Cell Death by SecTRAPs: Thioredoxin Reductase as a Prooxidant Killer of Cells

“…a,The specific activity was determined with the standard DTNB assay [50] using 10 nM enzyme preparation.b,Sec content was estimated from a combined assessment of specific activity, 75 Se incorporation, production and purification method and comparisons to earlier determinations [43]–[46], [49].c,Recombinant rat TrxR1 derivatized with cisplatin.d,Mutant C59S/C64S rat TrxR1 derivatized with cisplatin.…”

The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities

“…The DNA was then transformed into Escherichia coli BL21(DE3) competent cells, and the mutations were verified by DNA sequencing (GATC Biotech, Konstanz, Germany). For the TrxR variants that contain the C-terminal -Gly-Cys-SecGly motif (wild type, C59S, C64S, C59S/C64S, Y116I, and Y116T), the assistant plasmid pSUABC was co-transformed into the same host bacteria in order to increase selenoprotein yield as described previously (30).…”

A highly efficient form of the selenocysteine insertion sequence element in protozoan parasites and its use in mammalian cells

“…In addition, efficiency of Sec insertion into recombinant proteins is low, because the major products are often the truncated forms of selenoproteins. To overcome this problem, several methods have been proposed (33)(34)(35)(36), but none is fully satisfactory. Furthermore, some selenoproteins can be expressed only in eukaryotes because of unique posttranslational modifications.…”