Selenoprotein production and the biotechnological applications

Recombinant selenoprotein production in E.coli

Selenocysteine insertion in E.coli.

In order to comprehend heterologous selenoprotein synthesis in bacteria, it is first necessary to review the main aspects of the bacterial selenoprotein translation machinery. This machinery is highly complex, as has been revealed in great detail by August Böck and coworkers. The critical elements are summarized in this figure.

The selenocysteine-specific elongation factor, SELB, binds to a secondary structure in the mRNA, a Selenocysteine Insertion Sequence (SECIS) element, following the UGA codon encoding Sec insertion (instead of termination by release factor 2 as encoded by UGA in non-selenoprotein genes). SELB catalyzes Sec insertion using a selenocysteinylated selenocysteine-specific tRNA, the SelC gene product, which originally is charged with a seryl residue, but is converted to a selenocysteinyl moiety by the SelA gene product, selenocysteine synthase, using selenophosphate as selenium donor, formed from selenide by selenophosphate synthetase, the SelD gene product. All in all, is the factors needed for selenoprotein synthesis in E. coli are the four SelA - D gene products and a SELB-compatible SECIS structure in the mRNA.

Mammalian selenoprotein mRNA's also contain a SECIS element, but with other structural characteristics and they are positioned in the 3'-UTR. This is not compatible with the E. coli translation machinery - for that reason mammalian selenoproteins are not possible to directly express in E. coli, but will always lead to termination of translation at the UGA selenocysteine codon. However, by tailoring a selenoprotein gene to contain a bacterial-type SECIS element, this makes it possible to express the product in E. coli.

An example is shown in the next figure:

Bacterial SECIS element.

Expression of mammalian (rat) TrxR1 as a selenoprotein in E. coli.

By engineering an E. coli-type SECIS element in the gene for rat thioredoxin reductase 1, it became possible to express the protein as a selenoprotein recombinantly in the bacteria, which is illustrated in this figure. On the left-hand side are Commassie-stained gels whereas the right-hand figures are autoradiograms of the same gels, showing 75Se-selenite labeled products. "pET-TR" encoded TrxR1 without a SECIS element in the gene, pET-TRS contained a natural formate dehydrogenase-derived SECIS element, whereas pET-TRSTER contained a SECIS element encoding the TrxR1 carboxyterminus (see first figure, above). Note that the presence of a SECIS element was necessary for selenium incorporation, but that when present the reserve capacity of E. coli to synthesize selenoproteins is widely surpassing the normal synthesis under aerobic growth of the natural FDH-O selenoprotein (indicated in the figure).

The expression of rat TrxR1 was described in

High-level expression in Escherichia coli of selenocysteine-containing rat thioredoxin reductase utilizing gene fusions with engineered bacterial-type SECIS elements and co-expression with the selA, selB and selC genes.
Arnér E, Sarioglu H, Lottspeich F, Holmgren A, Böck A
J. Mol. Biol. 1999 Oct;292(5):1003-16

The method of heterologous selenoprotein production is further discussed in the review

Recombinant expression of mammalian selenocysteine-containing thioredoxin reductase and other selenoproteins in Escherichia coli.
Arnér E
Meth. Enzymol. 2002 ;347():226-35

It was further optimized in the study

Assessment of production conditions for efficient use of Escherichia coli in high-yield heterologous recombinant selenoprotein synthesis.
Rengby O, Johansson L, Carlson L, Serini E, Vlamis-Gardikas A, Kårsnäs P, et al
Appl. Environ. Microbiol. 2004 Sep;70(9):5159-67

Optimized condition for recombinant selenoprotein production to achieve high specificity and yield

Construction and verification of the ORaa strain.

The construction of a host strain enabling RF2 titration (ORaa) is schematically shown here. (A) Targeted E. coli genomic region, with the transcribed parts of the recJ and prfB/lysS operons shown as boxes. The positions of the H1 and H2 homologous regions are shown, and the frameshift in the RF2 open reading frame is indicated by an asterisk.

In the box, the nucleotide sequence of the relevant region of the prfB gene is shown in lowercase letters, with the translated initial part of RF2 also given in one-letter amino acid code. The additional thymidine in the gene, leading to an in-frame UGA frameshift site in the RF2 mRNA, is shown in parentheses.

The region constituting H1 corresponds to the nucleotides between the two number signs (#), while the H2 region is indicated by plus signs. Indicated by arrows are also the two sites where Primer1a and Primer1b annealed in the PCR 1 amplification and the restriction sites introduced by this primer pair. (B) Flow scheme, showing templates and primers used in PCR 1 to PCR 4 together with the ligation steps and schematic drawings depicting the resulting products or chromosomal replacements.

The PBAD promoter regions are indicated by hatched boxes. FRT, FLP recombinase target sequence.

Scheme of the turbidostatic culture system.

A turbidostatic culture system was established in order to maintain continuous addition of glucose, follow culture growth, and enable recombinant protein induction in the same culture but at different time points after glucose addition. As schematically shown, to a culture volume of 200 ml (oxygenated by a magnetic stirrer), fresh medium was added with a pump, using another pump for removal of culture set at the same speed. The pump flow (Q) was continuously adjusted to the bacterial growth, so that the OD600 was maintained at 0.4. For this, the OD600 was measured every 30 min, after which the turbidostatic culture was diluted with fresh medium to an OD600 of 0.4, with removal of volume so that the culture was kept at 200 ml. Between OD600 measurements, the pumps were used to maintain constant glucose addition. With Keto-Diabur-Test sticks it was verified that the glucose concentration was kept at high levels throughout the fermentation. Samples (5 ml) were taken at different time points and used for RF2 level determinations and IPTG addition, allowing 4 h of production of recombinant TrxR, which was subsequently analyzed together with RF2. The growth rate in the turbidostatic culture was determined using the equation given in the figure, resulting in a calculation of the percentage of change in biomass per time unit.

Figures taken from

Titration and conditional knockdown of the prfB gene in Escherichia coli: effects on growth and overproduction of the recombinant mammalian selenoprotein thioredoxin reductase.
Rengby O, Arnér E
Appl. Environ. Microbiol. 2007 Jan;73(2):432-41

Further reading:

Assessment of production conditions for efficient use of Escherichia coli in high-yield heterologous recombinant selenoprotein synthesis.
Rengby O, Johansson L, Carlson L, Serini E, Vlamis-Gardikas A, Kårsnäs P, et al
Appl. Environ. Microbiol. 2004 Sep;70(9):5159-67

Development of a Sel-tag

Development of a Sel-tag.

Inspired by the properties of the C-terminal tetrapeptide motif of mammalian TrxR, we have developed a redox active Sel-tag for use with recombinant proteins. We found that this Sel-tag could be used for one-step purification of the tagged protein, selenolate-targeted fluorescent labeling, as well as selenolate-tageted radiolabeling, with either selenium-75 or with the positron-emitter carbon-11. Further development of this technique is currently a highly prioritized issue in our laboratory.

The results were published in the launch issue of the new Nature Methods journal

Exploiting the 21st amino acid-purifying and labeling proteins by selenolate targeting.
Johansson L, Chen C, Thorell J, Fredriksson A, Stone-Elander S, Gafvelin G, et al
Nat. Methods 2004 Oct;1(1):61-6

A detailed protocol of utilizing this technique was published in the Nature Protocols journal

Tagging recombinant proteins with a Sel-tag for purification, labeling with electrophilic compounds or radiolabeling with 11C.
Cheng Q, Stone-Elander S, Arnér E
Nat Protoc 2006 ;1(2):604-13

Schematic illustration of dithiol and selenolthiol tag applications.

To explore the importance of selenocysteine in such applications, we here analyzed four different redox active C-terminal motifs, carrying either dithiol (Gly-Cys-Cys-Gly or Ser-Cys-Cys-Ser) or selenolthiol (Gly-Cys-Sec-Gly or Ser-Cys-Sec-Ser) motifs. Utilizing these different functional motifs with the same recombinant protein (Fel d 1), we could assess their relative reactivity and potential usefulness for biotechnological applications. Either dithiol or selenolthiol tetrapeptide tags on recombinant proteins can be efficiently used in one-step purification via PAO sepharose. In addition, the selenolthiol variants (but not the dithiol tags) were highly reactive in rapid labeling with electrophilic fluorescent or the short-lived positron emitting compound 11CH3I. As a unique feature for selenocysteine-containing proteins, the selenolthiol variants also permit methodologically simple in situ [75Se]-g-emitting radiolabeling through recombinant [75Se]-Selenocysteine incorporation.

This work was described in

Selenolthiol and dithiol C-terminal tetrapeptide motifs for one-step purification and labeling of recombinant proteins produced in E. coli.
Cheng Q, Johansson L, Thorell J, Fredriksson A, Samén E, Stone-Elander S, et al
Chembiochem 2006 Dec;7(12):1976-81

This is a close collaborative project with Professor Sharon Stone-Elander and her research group at Karolinska Pharmacy, Karolinska University Hospital; and the Department of Clinical Neuroscience, Karolinska Institutet.