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[20] Recombinant Expression of Mammalian Selenocysteine-Containing Thioredoxin Reductase and Other Selenoproteins in Escherichia coli By ELIAS S. J. ARNE´ R Introduction Heterologous expression of recombinant proteins in Escherichia coli for use in basic research studies, as therapeutics, in diagnostics, or as research reagents, is a much utilized technique in molecular biology with high protein yield and a well-characterized production system. Recombinant protein production will also play a significant role in the analysis of the vast number of candidate proteins (the “proteomes”) predicted from the many diverse ongoing genomic sequencing programs. Many organisms of all branches of life carry proteins that contain selenocysteine, the 21st amino acid. Examples of these selenoproteins are the three formate dehydrogenase isoenzymes of E. coli or the mammalian selenoprotein P, glutathione peroxidases, thyroid hormone deiodinases, and thioredoxin reductases; about 20 mammalian selenoproteins have been characterized.1–5 The list of newly discovered naturally occurring selenoproteins will continue to increase, either through database searches and predictions or as the result of experimental screening approaches.6–8 The selenoproteins have until more recently generally been excluded from conventional production based on recombinant expression in bacteria, because of highly species-specific machineries for cotranslational insertion of the reactive selenocysteine residue. Yet, if the species barriers are circumvented, the advantages and potential applications of targeted selenocysteine (Sec) insertion into recombinant proteins would include (1) use of conventional recombinant methodology for studies of naturally occurring selenoproteins, (2) the possibility of introducing selenocysteine into proteins for improving phase determination in X-ray crystallography or as a method for studying protein folding 1
E. S. J. Arn´er and A. Holmgren, Eur. J. Biochem. 267, 6102 (2000). A. B¨ock, K. Forchhammer, J. Heider, W. Leinfelder, G. Sawers, B. Veprek, and F. Zinoni, Mol. Microbiol. 5, 515 (1991). 3 A. H¨ uttenhofer and A. B¨ock, in “RNA Structure and Function” (R. W. Simons, and M. GrunbergManago, eds.), p. 603. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998. 4 S. C. Low and M. J. Berry, Trends Biochem. Sci. 21, 203 (1996). 5 T. C. Stadtman, Annu. Rev. Biochem. 65, 83 (1996). 6 A. Lescure, D. Gautheret, P. Carbon, and A. Krol, J. Biol. Chem. 274, 38147 (1999). 7 G. V. Kryukov, V. M. Kryukov, and V. N. Gladyshev, J. Biol. Chem. 274, 33888 (1999). 8 G. V. Kryukov and V. N. Gladyshev, Genes Cells 5, 1049 (2000). 2
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(reviewed in Besse et al.9 ), (3) targeting of Sec into proteins, making possible the insertion of high-energy selenium isotopes and constituting an alternative method of radiolabeling proteins with high specific activity, and (4) the possible use of selenium isotope-labeled proteins in positron emission tomography (PET) studies (discussed in Bergmann et al.10 ). The intricate synthesis machineries of selenoproteins in diverse organisms have been described in detail in several reviews2–5 as well as in other articles in this volume of Methods in Enzymology. The reader is therefore referred to those reviews and the mechanisms of selenoprotein synthesis shall not be repeated here at length. However, we should note that selenocysteine is not stable in its free form because of its high reactivity and the residue is in all organisms inserted cotranslationally at the position of an opal (UGA) codon, which normally confers termination of translation. The UGA codon is encoded as selenocysteine by highly complex translation machineries, which differ between gram-negative3,5 and gram-positive bacteria,11 archaea12 and higher eukaryotes.13–15 The synthesis machinery in E. coli has been characterized in detail mainly through the work of B¨ock and co-workers, using synthesis of formate dehydrogenase H as the main model system.2,3,5 The mRNA for the E. coli selenoprotein contains a specific sequence immediately following the UGA codon, with the nucleotides in this sequence both providing the information for translation of the amino acids following the selenocysteine residue, and forming a stem–loop secondary structure—a SECIS (selenocysteine insertion sequence) element. The SECIS element binds the SelB protein, the selB gene product. SelB is the selenocysteine-specific elongation factor, which is homologous to elongation factor EF-Tu but, in addition, has a carboxy-terminal domain that binds the SECIS element of the selenoprotein mRNA. SelB also binds a selenocysteine-specific tRNA (tRNASec), the selC gene product, in its selenocysteinylated form. In analogy with EF-Tu, SelB may then catalyze selenocysteine insertion at the ribosome, which occurs under hydrolysis of GTP and insertion of selenocysteine at the specific position of the selenocysteine-encoding UGA codon. The tRNASec is originally charged with a seryl residue which by selenocysteine synthase, the selA gene product, is converted to selenocysteinyl, utilizing selenophosphate as the selenium donor. The selenophosphate, in turn, is provided by 9
D. Besse, N. Budisa, W. Karnbrock, C. Minks, H. J. Musiol, S. Pegoraro, F. Siedler, E. Weyher, and L. Moroder, Biol. Chem. 378, 211 (1997). 10 R. Bergmann, P. Brust, G. Kampf, H. H. Coenen, and G. Stocklin, Nucl. Med. Biol. 22, 475 (1995). 11 T. Gursinsky, J. J¨ ager, J. R. Andreesen, and B. S¨ohling, Arch. Microbiol. 174, 200 (2000). 12 M. Rother, R. Wilting, S. Commans, and A. B¨ ock, J. Mol. Biol. 299, 351 (2000). 13 P. R. Copeland, V. A. Stepanik, and D. M. Driscoll, Mol. Cell. Biol. 21, 1491 (2001). 14 M. T. Nasim, S. Jaenecke, A. Belduz, H. Kollmus, L. Floh´ e, and J. E. McCarthy, J. Biol. Chem. 275, 14846 (2000). 15 D. Fagegaltier, N. Hubert, K. Yamada, T. Mizutani, P. Carbon, and A. Krol, EMBO J. 19, 4796 (2000).
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selenophosphate synthetase, the selD gene product. Taken together, selenocysteine insertion during selenoprotein translation in E. coli involves a SelB-compatible SECIS element next to the UGA codon in the selenoprotein mRNA, and the selA, selB, selC, and selD gene products. A SECIS element is also present in mammalian selenoprotein mRNA but has other secondary structural features than those found in E. coli and, moreover, is situated in the 3 untranslated region several hundred nucleotides downstream of the selenocysteine-encoding UGA.4,16,17 Also archaea18 and gram-positive bacteria11 have characteristic SECIS elements, with the latter being somewhat reminiscent of the E. coli SECIS element.11 It should therefore be emphasized that although the specific secondary structures of mRNA directing selenocysteine insertion may be called SECIS elements in all organisms, in analogy to the nomenclature of the mammalian system,4 the location and secondary structures of the different SECIS elements in diverse organisms are generally not similar to, or compatible with, each other. The differences between SECIS elements are the basis for species barriers in recombinant selenoprotein synthesis. In this article the critical factors for successfully by-passing the species barrier in order to enable heterologous selenoprotein production in E. coli are discussed with a focus on the possible general use of the technique. tRNASec Defining the Selenoprotein World The specific tRNA for selenocysteine insertion, tRNASec with it UCA anticodon, may be viewed as the common denominator of the selenoprotein world. Homozygous tRNASec-deficient mice display early embryonic death,19 indicating vital functions of one or several mammalian selenoproteins. With the number of canonical tRNAs identified in sequence-determined genomes ranging from 36 for Methanococcus jannaschii to 497 for humans or 584 for Caenorhabditis elegans it is interesting to note that these organisms (human, C. elegans, Drosophila melanogaster, M. Jannaschii, and E. coli) all seem to rely on a single tRNASec gene for selenoprotein synthesis.20 Moreover, as a common denominator, tRNASec from several species can complement an E. coli mutant deficient in tRNASec.11,21–23 16
˚ L. Zhong, E. S. J. Arn´er, J. Ljung, F. Aslund, and A. Holmgren, J. Biol. Chem. 273, 8581 (1998). R. Walczak, E. Westhof, P. Carbon, and A. Krol, RNA 2, 367 (1996). 18 M. Rother, A. Resch, W. L. Gardner, W. B. Whitman, and A. B¨ ock, Mol. Microbiol. 40, 900 (2001). 19 M. R. Bosl, K. Takaku, M. Oshima, S. Nishimura, and M. M. Taketo, Proc. Natl. Acad. Sci. U.S.A. 94, 5531 (1997). 20 International Human Genome Sequencing Consortium, Nature (London) 409, 860 (2001). 21 J. Heider, W. Leinfelder, and A. B¨ ock, Nucleic Acids Res. 17, 2529 (1989). 22 P. Tormay, R. Wilting, J. Heider, and A. B¨ ock, J. Bacteriol. 176, 1268 (1994). 23 C. Baron, C. Sturchler, X. Q. Wu, H. J. Gross, A. Krol, and A. B¨ ock, Nucleic Acids Res. 22, 228 (1994). 17
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As long as a tRNASec species is expressed at a balanced level, its function or specificity thereby is not generally a factor, making heterologous selenoprotein synthesis in E. coli a challenging task. Species Barriers in Heterologous Selenoprotein Synthesis It was recognized early on that the TGA in a mammalian glutathione peroxidase cDNA could not direct selenocysteine incorporation at the corresponding UGA when it was expressed in E. coli24 and the same incompatibility was found for glycine reductase of Clostridium sticklandii.25 Methanobacterium formicicum has a cysteine homolog of the selenocysteine-containing E. coli formate dehydrogenase H. In 1992, Heider and B¨ock used this gene to demonstrate successful targeted selenocysteine insertion in E. coli by changing the UGC, encoding cysteine in the native M. formicicum gene, to UGA and introducing an E. coli SECIS element from the homologous formate dehydrogenase gene immediately following the UGA.26 They had at that time identified features of the E. coli formate dehydrogenase SECIS element necessary for directing selenocysteine insertion,27,28 which was the basis for those studies. Details of the barriers to heterologous expression of a selenoprotein gene in bacteria were subsequently analyzed by Tormay and B¨ock, demonstrating that not only must the heterologous SECIS element be compatible with the host selenoprotein synthesis machinery; the barrier can also not be overcome by concomitantly introducing additional heterologous selenocysteinyl-tRNA and SelB, most likely due to incompatibility between the heterologous SelB and the E. coli ribosome, as demonstrated by expressing the hydV gene from Desulfomicrobium baculatum in E. coli.29 The general capacity for synthesizing recombinant selenoproteins in E. coli was, however, demonstrated in an overproduction of the endogenous E. coli formate dehydrogenase H.30 That study demonstrated that the bacteria indeed possessed an inherent capacity for high-level selenoprotein production, but the production was found only under anaerobic conditions. The reason for strict anaerobic expression should have depended on use of the endogenous formate dehydrogenase H promoter kept in the plasmid construct30 because subsequent findings show that the capacity for selenoprotein production in E. coli is indeed also efficient under aerobic conditions. 24
C. Rocher, C. Faucheu, F. Herve, C. Benicourt, and J. L. Lalanne, Gene 98, 193 (1991). G. E. Garcia and T. C. Stadtman, J. Bacteriol. 174, 7080 (1992). 26 J. Heider and A. B¨ ock, J. Bacteriol. 174, 659 (1992). 27 J. Heider, C. Baron, and A. B¨ ock, EMBO J. 11, 3759 (1992). 28 F. Zinoni, J. Heider, and A. B¨ ock, Proc. Natl. Acad. Sci. U.S.A. 87, 4660 (1990). 29 P. Tormay and A. B¨ ock, J. Bacteriol. 179, 576 (1997). 30 G. T. Chen, M. J. Axley, J. Hacia, and M. Inouye, Mol. Microbiol. 6, 781 (1992). 25
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Introduction of Selenocysteine Insertion Sequence Element Compatible with Bacterial Selenoprotein Synthesis Machinery as Method for Recombinant Selenoprotein Production When mammalian thioredoxin reductase was found to be a selenoprotein with a selenocysteine residue positioned in a unique carboxy-terminal -Gly-CysSec-Gly motif,16,31 we decided to analyze whether it was possible to engineer a bacterial-type SECIS element in the rat cDNA for the enzyme, thereby enabling targeted insertion of selenocysteine at the correct position in the recombinant selenoprotein. As is discussed below, the penultimate position of the Sec residue is particularly suitable for this strategy. The study, producing recombinant selenocysteine-containing rat thioredoxin reductase, showed that E. coli also has a high inherent capacity for selenoprotein production under aerobic conditions; this was the first evidence that the bacteria could be utilized for production of a recombinant mammalian selenoprotein.32 Other groups have since utilized the approach of engineering a bacterial-type SECIS element in a heterologous gene in order to enable expression in E. coli, demonstrating production of human thioredoxin reductase33,34 or of a cysteine to selenocysteine-substituted plant glutathione peroxidase.35 The critical factors in this production strategy are discussed in further detail below. Critical Factors for Recombinant Selenoprotein Production in Escherichia coli Functional Selenocysteine Insertion Sequence Element It is clear that the SECIS element that is to be introduced into a gene for expression as a selenoprotein in E. coli must be functionally compatible with the E. coli SelB elongation factor. The determinants for this interaction have been analyzed in detail by B¨ock and co-workers, as reviewed,3 and also further studied by others.35–39 To summarize, native E. coli SECIS elements are found in the three genes encoding the only selenoproteins of E. coli, namely formate dehydrogenase H (FDH-H, encoded by the fdhF gene), N ( fdnG), and O (being a formate 31
V. N. Gladyshev, K.-T. Jeang, and T. C. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 93, 6146 (1996). E. S. J. Arn´er, H. Sarioglu, F. Lottspeich, A. Holmgren, and A. B¨ock, J. Mol. Biol. 292, 1003 (1999). 33 S. Bar-Noy, S. N. Gorlatov, and T. C. Stadtman, Free Radic. Biol. Med. 30, 51 (2001). 34 R. Koishi, T. Nakamura, T. Takazawa, C. Yoshimura, and N. Serizawa, J. Biochem. (Tokyo) 127, 977 (2000). 35 S. Hazebrouck, L. Camoin, Z. Faltin, A. D. Strosberg, and Y. Eshdat, J. Biol. Chem. 275, 28715 (2000). 36 Z. Liu, M. Reches, and H. Engelberg-Kulka, J. Mol. Biol. 294, 1073 (1999). 37 Z. Liu, M. Reches, I. Groisman, and H. Engelberg-Kulka, Nucleic Acids Res. 26, 904 (1998). 38 K. E. Sandman and C. J. Noren, Nucleic Acids Res. 28, 755 (2000). 39 C. Li, M. Reches, and H. Engelberg-Kulka, J. Bacteriol. 182, 6302 (2000). 32
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oxidase and probably identical to FDH-Z encoded by fdoG40,41 ). Normally, FDH-H is expressed only under anaerobic conditions, FDH-N is induced anaerobically in the presence of nitrate, whereas the 110-kDa FDH-O is the only selenoprotein endogenously expressed in E. coli grown under aerobic conditions, during which it is expressed at low levels under conventional culture conditions.40 The SECIS elements of FDH-O and FDH-N are identical whereas that of FDH-H ( fdhF ) differs slightly from these, with the latter being the most studied in terms of the functional determinants for interaction with the SelB elongation factor. The major determinants of the fdhF SECIS necessary for maintained function as an SECIS element in E. coli involve preservation of the loop region binding SelB, which must be positioned at a distance of about 11 nucleotides downstream of the Sec UGA codon. These determinants are schematically shown in Fig. 1, where it is also shown how a variant of this SECIS element could be engineered to maintain function yet enable production of rat thioredoxin reductase when it was fused to the open reading frame of that enzyme and expressed in E. coli.32 SECIS elements that can be introduced into a gene tailored for heterologous selenoprotein production in E. coli other than those suggested from Fig. 1 may also be possible. Such novel SECIS elements may be characterized and found either by general screening of UGA suppression or function in SelB binding, using randomized sequence elements,42,43 or by combining the analysis of mutated SECIS elements with random mutagenesis of the SelB factor itself.39,44 Moreover, in the production of a selenocysteine-containing variant of plant glutathione peroxidase, it was reported that the proximal base pairs of the conserved loop region, around the bulged U nucleotide next to the loop (see Fig. 1), may possibly be substituted and still have maintained function.35 Use of such novel SECIS elements that deviate from the hitherto well-characterized consensus motifs (Fig. 1) in applications to achieve heterologous selenoprotein production should, however, be performed with great care to make certain that the supposedly improved system does not lead to suppression of the UGA codon by the insertion of other amino acids than selenocysteine (see also discussion on suppression below). Importance of Correct Stoichiometry The SelB elongation factor must form a quarternary complex with GTP, selenocysteinyl-tRNASec, and the SECIS element of the mRNA, and must interact 40
G. Sawers, J. Heider, E. Zehelein, and A. B¨ock, J. Bacteriol. 173, 4983 (1991). H. Abaibou, J. Pommier, S. Benoit, G. Giordano, and M. A. Mandrand-Berthelot, J. Bacteriol. 177, 7141 (1995). 42 S. J. Klug, A. H¨ uttenhofer, M. Kromayer, and M. Famulok, Proc. Natl. Acad. Sci. U.S.A. 94, 6676 (1997). 43 S. J. Klug, A. H¨ uttenhofer, and M. Famulok, RNA 5, 1180 (1999). 44 M. Kromayer, B. Neuhierl, A. Friebel, and A. B¨ ock, Mol. Gen. Genet. 262, 800 (1999). 41
H
OO
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with the ribosome in order to catalyze a completed peptidyltransferase reaction with insertion of selenocysteine.3 It is clear that the stoichiometry between the separate factors involved in this final step is of importance for resulting yield and efficiency in bacterial selenoprotein synthesis. Hence it was shown that sole overexpression of either SelB or tRNASec, or of an mRNA carrying an SECIS element, may reduce the UGA readthrough capacity as a result of disturbances in this stoichiometry.45 In essence, SelB may be tethered to nonfunctional binary or tertiary complexes with either tRNASec or the mRNA SECIS, thereby not forming the functional complete quarternary complex.3,45 We also found that the total yield of the selenocysteine-containing rat thioredoxin reductase expressed from a strong T7 promoter-driven pET vector could be increased about 8-fold (from 0.6 to 5 mg of selenoprotein produced per liter of E. coli culture) when the bacteria were cotransformed with a plasmid carrying accessory selA, selB, and selC genes under the control of their endogenous promoters.32 Use of titrable promoters guiding the expression level of the heterologous selenoprotein mRNA in order to achieve optimal stoichiometry and thereby total yield will possibly be shown as one method to further optimize this production system. Selenium Metabolism and Non-Selenocysteine-Mediated UGA Suppression Selenium is needed for selenoprotein production. However, selenium incorporation may also be unspecific, with selenium entering the cysteine or methionine pathways. This unspecific incorporation must be avoided in recombinant selenoprotein production and can be blocked by addition of excess L-cysteine or L-cystine to the growth medium.46 It is also important to note that an adequate selenium supply is essential for correct selenoprotein synthesis. In the absence of selenium in the growth medium the translation will either be terminated at the position of the
FIG. 1. Scheme of the determinants for a functional E. coli SECIS element. Top left: Native SECIS element of formate dehydrogenase H (encoded by the fdhF gene). The determinants for function in directing targeted cotranslational selenocysteine insertion at the UGA codon are indicated. The corresponding DNA sequence necessary to preserve these functional determinants is given below the SECIS element, in essence illustrating a SECIS cassette that may be introduced into a gene in order to achieve heterologous expression as a recombinant selenoprotein in E. coli. Top right: An example of such an engineered SECIS variant, which was utilized for production of rat thioredoxin reductase (TrxR), with the carboxy-terminal motif being -Gly-Cys-Sec-Gly-COOH [E. S. J. Arn´er, H. Sarioglu, F. Lottspeich, A. Holmgren, and A. B¨ock, J. Mol. Biol. 292, 1003 (1999)]. It should also be noted that the stem of the SECIS element may be shortened or elongated with a few nucleotides and yet maintain function [J. Heider, C. Baron, and A. B¨ock, EMBO J. 11, 3759 (1992)], and that the “stem” in fact does not need to base pair as long as the SelB-binding part is kept within a functional distance [Z. Liu, M. Reches, I. Groisman, and H. Engelberg-Kulka, Nucleic Acids Res. 26, 904 (1998)]. Below the DNA sequence corresponding to the SECIS determinants of the fdhF gene is also that of the slightly different fdnG gene, which probably also would be functional for use in heterologous selenoprotein production, although this has not yet been demonstrated. See text for further details.
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UGA or, more detrimental in the case of recombinant selenoprotein production, be suppressed by insertion of other amino acids than selenocysteine. It has been shown that the nucleotide immediately following the UGA codon may influence the extent of UGA suppression, with tryptophan most often suppressing the UGA when selenocysteine is not inserted,38 although the native SECIS element or the sequence immediately upstream of the UGA in fact seems to prevent tryptophan suppression.36 For practical applications in heterologous selenoprotein production it is important that as long as the selenium supply is sufficient and a proper SECIS element follows the UGA little or no non-selenocysteine-mediated UGA suppression can be noted.36,38 The resulting recombinant selenoprotein expressed under optimal conditions thereby either contains selenocysteine at the position of the UGA or, alternatively, becomes truncated at the site of the UGA. This was illustrated in the detailed characterization by mass spectrometry of tryptic digests of the rat thioredoxin reductase produced in E. coli, in which no peptide masses corresponding to non-selenocysteine-mediated UGA suppression could be detected.32 Inherent Lack of Efficiency in Bacterial Selenocysteine Insertion The UGA-directed selenocysteine insertion in E. coli is by nature inefficient, with a significant translational pause resulting from SelB binding to the SECIS element.47 This inefficiency may possibly affect the yield of recombinant selenoproteins produced in E. coli. Using the same plasmid carrying accessory selA, selB, and selC genes as utilized by us in the production of rat thioredoxin reductase (see above), the amount of full-length selenocysteine-containing product was found to be 11% (with 89% truncated protein formed) compared with 4% without overproduction of the accessory sel genes, when a native SECIS element was located within the open reading frame of an analyzed selenoprotein mRNA.47 This should be compared with the production of thioredoxin reductase, where termination codons were introduced into the nonconserved stem of the SECIS element in order to encode the carboxy-terminal -Sec-Gly-COOH motif of the enzyme (Fig. 1), resulting in about 25% full-length product using the accessory sel genes, compared with about 3% without such cotransformation.32 It is possible that this case, which in essence located the SelB-binding motif to an untranslated region of the mRNA (Fig. 1), increased the yield as a result of a less pronounced competition between SelB and EF-Tu. Inefficiencies in targeted selenocysteine insertion within an open reading frame may also be one of several possible factors leading to a low yield in heterologous production of the Sec-containing plant35 or human (our unpublished observations, 2000) glutathione peroxidases, using this technique. The 45
P. Tormay, A. Sawers, and A. B¨ock, Mol. Microbiol. 21, 1253 (1996). S. Muller, J. Heider, and A. B¨ock, Arch. Microbiol. 168, 421 (1997). 47 S. Suppmann, B. C. Persson, and A. B¨ ock, EMBO J. 18, 2284 (1999). 46
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positioning of a bacterial-type SECIS element within an open reading frame also imposes limitations to the use of codons corresponding to the SelB-binding loop region, which must be conserved (Fig. 1). Even if these limitations exist, the strategy nonetheless can also yield functional recombinant enzyme species when the selenocysteine residue is located at internal positions of a protein and the whole SECIS element therefore must be translated.35 Other Factors Influencing Bacterial Recombinant Protein Production Naturally, all the factors affecting yield in heterologous expression of recombinant proteins in E. coli, such as promoter or codon usage, growth conditions, inclusion body formation, proteolysis, and mRNA stability,48 also apply to production of recombinant selenoproteins. Such other factors, not depending on the fact that a selenoprotein is produced, may explain the lower yield reported for production of human thioredoxin reductases33,34 compared with the production of the corresponding enzyme from rat.32 Conclusions Escherichia coli has a significant capacity for heterologous production of recombinant selenoproteins, far exceeding the natural level of synthesis of the endogenous bacterial selenoproteins. Targeted insertion of selenocysteine at specific UGA codons in recombinant proteins requires, however, that attention be given to a number of critical factors as summarized herein. This production system may nonetheless be utilized in studies of naturally occuring selenoproteins as well as for the production of selenocysteine-containing variants of nonselenoproteins; the applications of such selenosubstituted species may be several, considering the high-energy irradiation of selenium isotopes that can be introduced or the specific physical and biochemical characteristics of the selenocysteine moiety.
48
G. Hannig and S. C. Makrides, Trends Biotechnol. 16, 54 (1998).
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[20] Recombinant Expression of Mammalian Selenocysteine-Containing Thioredoxin Reductase and Other Selenoproteins in Escherichia coli By ELIAS S. J. ARNE´ R Introduction Heterologous expression of recombinant proteins in Escherichia coli for use in basic research studies, as therapeutics, in diagnostics, or as research reagents, is a much utilized technique in molecular biology with high protein yield and a well-characterized production system. Recombinant protein production will also play a significant role in the analysis of the vast number of candidate proteins (the “proteomes”) predicted from the many diverse ongoing genomic sequencing programs. Many organisms of all branches of life carry proteins that contain selenocysteine, the 21st amino acid. Examples of these selenoproteins are the three formate dehydrogenase isoenzymes of E. coli or the mammalian selenoprotein P, glutathione peroxidases, thyroid hormone deiodinases, and thioredoxin reductases; about 20 mammalian selenoproteins have been characterized.1–5 The list of newly discovered naturally occurring selenoproteins will continue to increase, either through database searches and predictions or as the result of experimental screening approaches.6–8 The selenoproteins have until more recently generally been excluded from conventional production based on recombinant expression in bacteria, because of highly species-specific machineries for cotranslational insertion of the reactive selenocysteine residue. Yet, if the species barriers are circumvented, the advantages and potential applications of targeted selenocysteine (Sec) insertion into recombinant proteins would include (1) use of conventional recombinant methodology for studies of naturally occurring selenoproteins, (2) the possibility of introducing selenocysteine into proteins for improving phase determination in X-ray crystallography or as a method for studying protein folding 1
E. S. J. Arn´er and A. Holmgren, Eur. J. Biochem. 267, 6102 (2000). A. B¨ock, K. Forchhammer, J. Heider, W. Leinfelder, G. Sawers, B. Veprek, and F. Zinoni, Mol. Microbiol. 5, 515 (1991). 3 A. H¨ uttenhofer and A. B¨ock, in “RNA Structure and Function” (R. W. Simons, and M. GrunbergManago, eds.), p. 603. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998. 4 S. C. Low and M. J. Berry, Trends Biochem. Sci. 21, 203 (1996). 5 T. C. Stadtman, Annu. Rev. Biochem. 65, 83 (1996). 6 A. Lescure, D. Gautheret, P. Carbon, and A. Krol, J. Biol. Chem. 274, 38147 (1999). 7 G. V. Kryukov, V. M. Kryukov, and V. N. Gladyshev, J. Biol. Chem. 274, 33888 (1999). 8 G. V. Kryukov and V. N. Gladyshev, Genes Cells 5, 1049 (2000). 2
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(reviewed in Besse et al.9 ), (3) targeting of Sec into proteins, making possible the insertion of high-energy selenium isotopes and constituting an alternative method of radiolabeling proteins with high specific activity, and (4) the possible use of selenium isotope-labeled proteins in positron emission tomography (PET) studies (discussed in Bergmann et al.10 ). The intricate synthesis machineries of selenoproteins in diverse organisms have been described in detail in several reviews2–5 as well as in other articles in this volume of Methods in Enzymology. The reader is therefore referred to those reviews and the mechanisms of selenoprotein synthesis shall not be repeated here at length. However, we should note that selenocysteine is not stable in its free form because of its high reactivity and the residue is in all organisms inserted cotranslationally at the position of an opal (UGA) codon, which normally confers termination of translation. The UGA codon is encoded as selenocysteine by highly complex translation machineries, which differ between gram-negative3,5 and gram-positive bacteria,11 archaea12 and higher eukaryotes.13–15 The synthesis machinery in E. coli has been characterized in detail mainly through the work of B¨ock and co-workers, using synthesis of formate dehydrogenase H as the main model system.2,3,5 The mRNA for the E. coli selenoprotein contains a specific sequence immediately following the UGA codon, with the nucleotides in this sequence both providing the information for translation of the amino acids following the selenocysteine residue, and forming a stem–loop secondary structure—a SECIS (selenocysteine insertion sequence) element. The SECIS element binds the SelB protein, the selB gene product. SelB is the selenocysteine-specific elongation factor, which is homologous to elongation factor EF-Tu but, in addition, has a carboxy-terminal domain that binds the SECIS element of the selenoprotein mRNA. SelB also binds a selenocysteine-specific tRNA (tRNASec), the selC gene product, in its selenocysteinylated form. In analogy with EF-Tu, SelB may then catalyze selenocysteine insertion at the ribosome, which occurs under hydrolysis of GTP and insertion of selenocysteine at the specific position of the selenocysteine-encoding UGA codon. The tRNASec is originally charged with a seryl residue which by selenocysteine synthase, the selA gene product, is converted to selenocysteinyl, utilizing selenophosphate as the selenium donor. The selenophosphate, in turn, is provided by 9
D. Besse, N. Budisa, W. Karnbrock, C. Minks, H. J. Musiol, S. Pegoraro, F. Siedler, E. Weyher, and L. Moroder, Biol. Chem. 378, 211 (1997). 10 R. Bergmann, P. Brust, G. Kampf, H. H. Coenen, and G. Stocklin, Nucl. Med. Biol. 22, 475 (1995). 11 T. Gursinsky, J. J¨ ager, J. R. Andreesen, and B. S¨ohling, Arch. Microbiol. 174, 200 (2000). 12 M. Rother, R. Wilting, S. Commans, and A. B¨ ock, J. Mol. Biol. 299, 351 (2000). 13 P. R. Copeland, V. A. Stepanik, and D. M. Driscoll, Mol. Cell. Biol. 21, 1491 (2001). 14 M. T. Nasim, S. Jaenecke, A. Belduz, H. Kollmus, L. Floh´ e, and J. E. McCarthy, J. Biol. Chem. 275, 14846 (2000). 15 D. Fagegaltier, N. Hubert, K. Yamada, T. Mizutani, P. Carbon, and A. Krol, EMBO J. 19, 4796 (2000).
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selenophosphate synthetase, the selD gene product. Taken together, selenocysteine insertion during selenoprotein translation in E. coli involves a SelB-compatible SECIS element next to the UGA codon in the selenoprotein mRNA, and the selA, selB, selC, and selD gene products. A SECIS element is also present in mammalian selenoprotein mRNA but has other secondary structural features than those found in E. coli and, moreover, is situated in the 3 untranslated region several hundred nucleotides downstream of the selenocysteine-encoding UGA.4,16,17 Also archaea18 and gram-positive bacteria11 have characteristic SECIS elements, with the latter being somewhat reminiscent of the E. coli SECIS element.11 It should therefore be emphasized that although the specific secondary structures of mRNA directing selenocysteine insertion may be called SECIS elements in all organisms, in analogy to the nomenclature of the mammalian system,4 the location and secondary structures of the different SECIS elements in diverse organisms are generally not similar to, or compatible with, each other. The differences between SECIS elements are the basis for species barriers in recombinant selenoprotein synthesis. In this article the critical factors for successfully by-passing the species barrier in order to enable heterologous selenoprotein production in E. coli are discussed with a focus on the possible general use of the technique. tRNASec Defining the Selenoprotein World The specific tRNA for selenocysteine insertion, tRNASec with it UCA anticodon, may be viewed as the common denominator of the selenoprotein world. Homozygous tRNASec-deficient mice display early embryonic death,19 indicating vital functions of one or several mammalian selenoproteins. With the number of canonical tRNAs identified in sequence-determined genomes ranging from 36 for Methanococcus jannaschii to 497 for humans or 584 for Caenorhabditis elegans it is interesting to note that these organisms (human, C. elegans, Drosophila melanogaster, M. Jannaschii, and E. coli) all seem to rely on a single tRNASec gene for selenoprotein synthesis.20 Moreover, as a common denominator, tRNASec from several species can complement an E. coli mutant deficient in tRNASec.11,21–23 16
˚ L. Zhong, E. S. J. Arn´er, J. Ljung, F. Aslund, and A. Holmgren, J. Biol. Chem. 273, 8581 (1998). R. Walczak, E. Westhof, P. Carbon, and A. Krol, RNA 2, 367 (1996). 18 M. Rother, A. Resch, W. L. Gardner, W. B. Whitman, and A. B¨ ock, Mol. Microbiol. 40, 900 (2001). 19 M. R. Bosl, K. Takaku, M. Oshima, S. Nishimura, and M. M. Taketo, Proc. Natl. Acad. Sci. U.S.A. 94, 5531 (1997). 20 International Human Genome Sequencing Consortium, Nature (London) 409, 860 (2001). 21 J. Heider, W. Leinfelder, and A. B¨ ock, Nucleic Acids Res. 17, 2529 (1989). 22 P. Tormay, R. Wilting, J. Heider, and A. B¨ ock, J. Bacteriol. 176, 1268 (1994). 23 C. Baron, C. Sturchler, X. Q. Wu, H. J. Gross, A. Krol, and A. B¨ ock, Nucleic Acids Res. 22, 228 (1994). 17
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As long as a tRNASec species is expressed at a balanced level, its function or specificity thereby is not generally a factor, making heterologous selenoprotein synthesis in E. coli a challenging task. Species Barriers in Heterologous Selenoprotein Synthesis It was recognized early on that the TGA in a mammalian glutathione peroxidase cDNA could not direct selenocysteine incorporation at the corresponding UGA when it was expressed in E. coli24 and the same incompatibility was found for glycine reductase of Clostridium sticklandii.25 Methanobacterium formicicum has a cysteine homolog of the selenocysteine-containing E. coli formate dehydrogenase H. In 1992, Heider and B¨ock used this gene to demonstrate successful targeted selenocysteine insertion in E. coli by changing the UGC, encoding cysteine in the native M. formicicum gene, to UGA and introducing an E. coli SECIS element from the homologous formate dehydrogenase gene immediately following the UGA.26 They had at that time identified features of the E. coli formate dehydrogenase SECIS element necessary for directing selenocysteine insertion,27,28 which was the basis for those studies. Details of the barriers to heterologous expression of a selenoprotein gene in bacteria were subsequently analyzed by Tormay and B¨ock, demonstrating that not only must the heterologous SECIS element be compatible with the host selenoprotein synthesis machinery; the barrier can also not be overcome by concomitantly introducing additional heterologous selenocysteinyl-tRNA and SelB, most likely due to incompatibility between the heterologous SelB and the E. coli ribosome, as demonstrated by expressing the hydV gene from Desulfomicrobium baculatum in E. coli.29 The general capacity for synthesizing recombinant selenoproteins in E. coli was, however, demonstrated in an overproduction of the endogenous E. coli formate dehydrogenase H.30 That study demonstrated that the bacteria indeed possessed an inherent capacity for high-level selenoprotein production, but the production was found only under anaerobic conditions. The reason for strict anaerobic expression should have depended on use of the endogenous formate dehydrogenase H promoter kept in the plasmid construct30 because subsequent findings show that the capacity for selenoprotein production in E. coli is indeed also efficient under aerobic conditions. 24
C. Rocher, C. Faucheu, F. Herve, C. Benicourt, and J. L. Lalanne, Gene 98, 193 (1991). G. E. Garcia and T. C. Stadtman, J. Bacteriol. 174, 7080 (1992). 26 J. Heider and A. B¨ ock, J. Bacteriol. 174, 659 (1992). 27 J. Heider, C. Baron, and A. B¨ ock, EMBO J. 11, 3759 (1992). 28 F. Zinoni, J. Heider, and A. B¨ ock, Proc. Natl. Acad. Sci. U.S.A. 87, 4660 (1990). 29 P. Tormay and A. B¨ ock, J. Bacteriol. 179, 576 (1997). 30 G. T. Chen, M. J. Axley, J. Hacia, and M. Inouye, Mol. Microbiol. 6, 781 (1992). 25
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Introduction of Selenocysteine Insertion Sequence Element Compatible with Bacterial Selenoprotein Synthesis Machinery as Method for Recombinant Selenoprotein Production When mammalian thioredoxin reductase was found to be a selenoprotein with a selenocysteine residue positioned in a unique carboxy-terminal -Gly-CysSec-Gly motif,16,31 we decided to analyze whether it was possible to engineer a bacterial-type SECIS element in the rat cDNA for the enzyme, thereby enabling targeted insertion of selenocysteine at the correct position in the recombinant selenoprotein. As is discussed below, the penultimate position of the Sec residue is particularly suitable for this strategy. The study, producing recombinant selenocysteine-containing rat thioredoxin reductase, showed that E. coli also has a high inherent capacity for selenoprotein production under aerobic conditions; this was the first evidence that the bacteria could be utilized for production of a recombinant mammalian selenoprotein.32 Other groups have since utilized the approach of engineering a bacterial-type SECIS element in a heterologous gene in order to enable expression in E. coli, demonstrating production of human thioredoxin reductase33,34 or of a cysteine to selenocysteine-substituted plant glutathione peroxidase.35 The critical factors in this production strategy are discussed in further detail below. Critical Factors for Recombinant Selenoprotein Production in Escherichia coli Functional Selenocysteine Insertion Sequence Element It is clear that the SECIS element that is to be introduced into a gene for expression as a selenoprotein in E. coli must be functionally compatible with the E. coli SelB elongation factor. The determinants for this interaction have been analyzed in detail by B¨ock and co-workers, as reviewed,3 and also further studied by others.35–39 To summarize, native E. coli SECIS elements are found in the three genes encoding the only selenoproteins of E. coli, namely formate dehydrogenase H (FDH-H, encoded by the fdhF gene), N ( fdnG), and O (being a formate 31
V. N. Gladyshev, K.-T. Jeang, and T. C. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 93, 6146 (1996). E. S. J. Arn´er, H. Sarioglu, F. Lottspeich, A. Holmgren, and A. B¨ock, J. Mol. Biol. 292, 1003 (1999). 33 S. Bar-Noy, S. N. Gorlatov, and T. C. Stadtman, Free Radic. Biol. Med. 30, 51 (2001). 34 R. Koishi, T. Nakamura, T. Takazawa, C. Yoshimura, and N. Serizawa, J. Biochem. (Tokyo) 127, 977 (2000). 35 S. Hazebrouck, L. Camoin, Z. Faltin, A. D. Strosberg, and Y. Eshdat, J. Biol. Chem. 275, 28715 (2000). 36 Z. Liu, M. Reches, and H. Engelberg-Kulka, J. Mol. Biol. 294, 1073 (1999). 37 Z. Liu, M. Reches, I. Groisman, and H. Engelberg-Kulka, Nucleic Acids Res. 26, 904 (1998). 38 K. E. Sandman and C. J. Noren, Nucleic Acids Res. 28, 755 (2000). 39 C. Li, M. Reches, and H. Engelberg-Kulka, J. Bacteriol. 182, 6302 (2000). 32
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oxidase and probably identical to FDH-Z encoded by fdoG40,41 ). Normally, FDH-H is expressed only under anaerobic conditions, FDH-N is induced anaerobically in the presence of nitrate, whereas the 110-kDa FDH-O is the only selenoprotein endogenously expressed in E. coli grown under aerobic conditions, during which it is expressed at low levels under conventional culture conditions.40 The SECIS elements of FDH-O and FDH-N are identical whereas that of FDH-H ( fdhF ) differs slightly from these, with the latter being the most studied in terms of the functional determinants for interaction with the SelB elongation factor. The major determinants of the fdhF SECIS necessary for maintained function as an SECIS element in E. coli involve preservation of the loop region binding SelB, which must be positioned at a distance of about 11 nucleotides downstream of the Sec UGA codon. These determinants are schematically shown in Fig. 1, where it is also shown how a variant of this SECIS element could be engineered to maintain function yet enable production of rat thioredoxin reductase when it was fused to the open reading frame of that enzyme and expressed in E. coli.32 SECIS elements that can be introduced into a gene tailored for heterologous selenoprotein production in E. coli other than those suggested from Fig. 1 may also be possible. Such novel SECIS elements may be characterized and found either by general screening of UGA suppression or function in SelB binding, using randomized sequence elements,42,43 or by combining the analysis of mutated SECIS elements with random mutagenesis of the SelB factor itself.39,44 Moreover, in the production of a selenocysteine-containing variant of plant glutathione peroxidase, it was reported that the proximal base pairs of the conserved loop region, around the bulged U nucleotide next to the loop (see Fig. 1), may possibly be substituted and still have maintained function.35 Use of such novel SECIS elements that deviate from the hitherto well-characterized consensus motifs (Fig. 1) in applications to achieve heterologous selenoprotein production should, however, be performed with great care to make certain that the supposedly improved system does not lead to suppression of the UGA codon by the insertion of other amino acids than selenocysteine (see also discussion on suppression below). Importance of Correct Stoichiometry The SelB elongation factor must form a quarternary complex with GTP, selenocysteinyl-tRNASec, and the SECIS element of the mRNA, and must interact 40
G. Sawers, J. Heider, E. Zehelein, and A. B¨ock, J. Bacteriol. 173, 4983 (1991). H. Abaibou, J. Pommier, S. Benoit, G. Giordano, and M. A. Mandrand-Berthelot, J. Bacteriol. 177, 7141 (1995). 42 S. J. Klug, A. H¨ uttenhofer, M. Kromayer, and M. Famulok, Proc. Natl. Acad. Sci. U.S.A. 94, 6676 (1997). 43 S. J. Klug, A. H¨ uttenhofer, and M. Famulok, RNA 5, 1180 (1999). 44 M. Kromayer, B. Neuhierl, A. Friebel, and A. B¨ ock, Mol. Gen. Genet. 262, 800 (1999). 41
H
OO
y-C
-Gl
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ys-
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with the ribosome in order to catalyze a completed peptidyltransferase reaction with insertion of selenocysteine.3 It is clear that the stoichiometry between the separate factors involved in this final step is of importance for resulting yield and efficiency in bacterial selenoprotein synthesis. Hence it was shown that sole overexpression of either SelB or tRNASec, or of an mRNA carrying an SECIS element, may reduce the UGA readthrough capacity as a result of disturbances in this stoichiometry.45 In essence, SelB may be tethered to nonfunctional binary or tertiary complexes with either tRNASec or the mRNA SECIS, thereby not forming the functional complete quarternary complex.3,45 We also found that the total yield of the selenocysteine-containing rat thioredoxin reductase expressed from a strong T7 promoter-driven pET vector could be increased about 8-fold (from 0.6 to 5 mg of selenoprotein produced per liter of E. coli culture) when the bacteria were cotransformed with a plasmid carrying accessory selA, selB, and selC genes under the control of their endogenous promoters.32 Use of titrable promoters guiding the expression level of the heterologous selenoprotein mRNA in order to achieve optimal stoichiometry and thereby total yield will possibly be shown as one method to further optimize this production system. Selenium Metabolism and Non-Selenocysteine-Mediated UGA Suppression Selenium is needed for selenoprotein production. However, selenium incorporation may also be unspecific, with selenium entering the cysteine or methionine pathways. This unspecific incorporation must be avoided in recombinant selenoprotein production and can be blocked by addition of excess L-cysteine or L-cystine to the growth medium.46 It is also important to note that an adequate selenium supply is essential for correct selenoprotein synthesis. In the absence of selenium in the growth medium the translation will either be terminated at the position of the
FIG. 1. Scheme of the determinants for a functional E. coli SECIS element. Top left: Native SECIS element of formate dehydrogenase H (encoded by the fdhF gene). The determinants for function in directing targeted cotranslational selenocysteine insertion at the UGA codon are indicated. The corresponding DNA sequence necessary to preserve these functional determinants is given below the SECIS element, in essence illustrating a SECIS cassette that may be introduced into a gene in order to achieve heterologous expression as a recombinant selenoprotein in E. coli. Top right: An example of such an engineered SECIS variant, which was utilized for production of rat thioredoxin reductase (TrxR), with the carboxy-terminal motif being -Gly-Cys-Sec-Gly-COOH [E. S. J. Arn´er, H. Sarioglu, F. Lottspeich, A. Holmgren, and A. B¨ock, J. Mol. Biol. 292, 1003 (1999)]. It should also be noted that the stem of the SECIS element may be shortened or elongated with a few nucleotides and yet maintain function [J. Heider, C. Baron, and A. B¨ock, EMBO J. 11, 3759 (1992)], and that the “stem” in fact does not need to base pair as long as the SelB-binding part is kept within a functional distance [Z. Liu, M. Reches, I. Groisman, and H. Engelberg-Kulka, Nucleic Acids Res. 26, 904 (1998)]. Below the DNA sequence corresponding to the SECIS determinants of the fdhF gene is also that of the slightly different fdnG gene, which probably also would be functional for use in heterologous selenoprotein production, although this has not yet been demonstrated. See text for further details.
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UGA or, more detrimental in the case of recombinant selenoprotein production, be suppressed by insertion of other amino acids than selenocysteine. It has been shown that the nucleotide immediately following the UGA codon may influence the extent of UGA suppression, with tryptophan most often suppressing the UGA when selenocysteine is not inserted,38 although the native SECIS element or the sequence immediately upstream of the UGA in fact seems to prevent tryptophan suppression.36 For practical applications in heterologous selenoprotein production it is important that as long as the selenium supply is sufficient and a proper SECIS element follows the UGA little or no non-selenocysteine-mediated UGA suppression can be noted.36,38 The resulting recombinant selenoprotein expressed under optimal conditions thereby either contains selenocysteine at the position of the UGA or, alternatively, becomes truncated at the site of the UGA. This was illustrated in the detailed characterization by mass spectrometry of tryptic digests of the rat thioredoxin reductase produced in E. coli, in which no peptide masses corresponding to non-selenocysteine-mediated UGA suppression could be detected.32 Inherent Lack of Efficiency in Bacterial Selenocysteine Insertion The UGA-directed selenocysteine insertion in E. coli is by nature inefficient, with a significant translational pause resulting from SelB binding to the SECIS element.47 This inefficiency may possibly affect the yield of recombinant selenoproteins produced in E. coli. Using the same plasmid carrying accessory selA, selB, and selC genes as utilized by us in the production of rat thioredoxin reductase (see above), the amount of full-length selenocysteine-containing product was found to be 11% (with 89% truncated protein formed) compared with 4% without overproduction of the accessory sel genes, when a native SECIS element was located within the open reading frame of an analyzed selenoprotein mRNA.47 This should be compared with the production of thioredoxin reductase, where termination codons were introduced into the nonconserved stem of the SECIS element in order to encode the carboxy-terminal -Sec-Gly-COOH motif of the enzyme (Fig. 1), resulting in about 25% full-length product using the accessory sel genes, compared with about 3% without such cotransformation.32 It is possible that this case, which in essence located the SelB-binding motif to an untranslated region of the mRNA (Fig. 1), increased the yield as a result of a less pronounced competition between SelB and EF-Tu. Inefficiencies in targeted selenocysteine insertion within an open reading frame may also be one of several possible factors leading to a low yield in heterologous production of the Sec-containing plant35 or human (our unpublished observations, 2000) glutathione peroxidases, using this technique. The 45
P. Tormay, A. Sawers, and A. B¨ock, Mol. Microbiol. 21, 1253 (1996). S. Muller, J. Heider, and A. B¨ock, Arch. Microbiol. 168, 421 (1997). 47 S. Suppmann, B. C. Persson, and A. B¨ ock, EMBO J. 18, 2284 (1999). 46
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positioning of a bacterial-type SECIS element within an open reading frame also imposes limitations to the use of codons corresponding to the SelB-binding loop region, which must be conserved (Fig. 1). Even if these limitations exist, the strategy nonetheless can also yield functional recombinant enzyme species when the selenocysteine residue is located at internal positions of a protein and the whole SECIS element therefore must be translated.35 Other Factors Influencing Bacterial Recombinant Protein Production Naturally, all the factors affecting yield in heterologous expression of recombinant proteins in E. coli, such as promoter or codon usage, growth conditions, inclusion body formation, proteolysis, and mRNA stability,48 also apply to production of recombinant selenoproteins. Such other factors, not depending on the fact that a selenoprotein is produced, may explain the lower yield reported for production of human thioredoxin reductases33,34 compared with the production of the corresponding enzyme from rat.32 Conclusions Escherichia coli has a significant capacity for heterologous production of recombinant selenoproteins, far exceeding the natural level of synthesis of the endogenous bacterial selenoproteins. Targeted insertion of selenocysteine at specific UGA codons in recombinant proteins requires, however, that attention be given to a number of critical factors as summarized herein. This production system may nonetheless be utilized in studies of naturally occuring selenoproteins as well as for the production of selenocysteine-containing variants of nonselenoproteins; the applications of such selenosubstituted species may be several, considering the high-energy irradiation of selenium isotopes that can be introduced or the specific physical and biochemical characteristics of the selenocysteine moiety.
48
G. Hannig and S. C. Makrides, Trends Biotechnol. 16, 54 (1998).