Custom selenoprotein production enabled by laboratory evolution of recoded bacterial strains

Abstract

Incorporation of the rare amino acid selenocysteine to form diselenide bonds can improve stability and function of synthetic peptide therapeutics. However, application of this approach to recombinant proteins has been hampered by heterogeneous incorporation, low selenoprotein yields, and poor fitness of bacterial producer strains. We report the evolution of recoded Escherichia coli strains with improved fitness that are superior hosts for recombinant selenoprotein production. We apply an engineered β-lactamase containing an essential diselenide bond to enforce selenocysteine dependence during continuous evolution of recoded E. coli strains. Evolved strains maintain an expanded genetic code indefinitely. We engineer a fluorescent reporter to quantify selenocysteine incorporation in vivo and show complete decoding of UAG codons as selenocysteine. Replacement of native, labile disulfide bonds in antibody fragments with diselenide bonds vastly improves resistance to reducing conditions. Highly seleno-competent bacterial strains enable industrial-scale selenoprotein expression and unique diselenide architecture, advancing our ability to customize the selenoproteome.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Evolution of selenocysteine-dependent E. coli strains improves fitness.
Figure 2: Genes mutated during continuous evolution.
Figure 3: Selenocysteine incorporation in evolved strains.
Figure 4: Recombinant seleno-scFvs containing diselenide bonds.
Figure 5: MS and ELISA curves for anti-RCA scFvs.

Accession codes

Primary accessions

NCBI Reference Sequence

Referenced accessions

Protein Data Bank

References

  1. 1

    Yoshizawa, S. & Böck, A. The many levels of control on bacterial selenoprotein synthesis. Biochim. Biophys. Acta 1790, 1404–1414 (2009).

    CAS  PubMed  Article  Google Scholar 

  2. 2

    Suppmann, S., Persson, B.C. & Böck, A. Dynamics and efficiency in vivo of UGA-directed selenocysteine insertion at the ribosome. EMBO J. 18, 2284–2293 (1999).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. 3

    Arnér, E.S., Sarioglu, H., Lottspeich, F., Holmgren, A. & Böck, A. 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. J. Mol. Biol. 292, 1003–1016 (1999).

    PubMed  Article  Google Scholar 

  4. 4

    Thyer, R., Filipovska, A. & Rackham, O. Engineered rRNA enhances the efficiency of selenocysteine incorporation during translation. J. Am. Chem. Soc. 135, 2–5 (2013).

    CAS  PubMed  Article  Google Scholar 

  5. 5

    Thyer, R., Robotham, S.A., Brodbelt, J.S. & Ellington, A.D. Evolving tRNA(Sec) for efficient canonical incorporation of selenocysteine. J. Am. Chem. Soc. 137, 46–49 (2015).

    CAS  PubMed  Article  Google Scholar 

  6. 6

    Lajoie, M.J. et al. Genomically recoded organisms expand biological functions. Science 342, 357–360 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7

    Blount, Z.D., Barrick, J.E., Davidson, C.J. & Lenski, R.E. Genomic analysis of a key innovation in an experimental Escherichia coli population. Nature 489, 513–518 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. 8

    Quandt, E.M. et al. Fine-tuning citrate synthase flux potentiates and refines metabolic innovation in the Lenski evolution experiment. eLife 4, 09696 (2015).

    Article  Google Scholar 

  9. 9

    Majiduddin, F.K. & Palzkill, T. Amino acid sequence requirements at residues 69 and 238 for the SME-1 beta-lactamase to confer resistance to beta-lactam antibiotics. Antimicrob. Agents Chemother. 47, 1062–1067 (2003).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. 10

    Gutierrez, A. β-Lactam antibiotics promote bacterial mutagenesis via an RpoS-mediated reduction in replication fidelity. Nat. Commun. 4, 1610 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. 11

    Layton, J.C. & Foster, P.L. Error-prone DNA polymerase IV is regulated by the heat shock chaperone GroE in Escherichia coli. J. Bacteriol. 187, 449–457 (2005).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. 12

    Ishii, T.M., Kotlova, N., Tapsoba, F. & Steinberg, S.V. The long D-stem of the selenocysteine tRNA provides resilience at the expense of maximal function. J. Biol. Chem. 288, 13337–13344 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. 13

    Rudolph, B., Gebendorfer, K.M., Buchner, J. & Winter, J. Evolution of Escherichia coli for growth at high temperatures. J. Biol. Chem. 285, 19029–19034 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14

    Nilsson, M. & Rydén-Aulin, M. Glutamine is incorporated at the nonsense codons UAG and UAA in a suppressor-free Escherichia coli strain. Biochim. Biophys. Acta 1627, 1–6 (2003).

    CAS  PubMed  Article  Google Scholar 

  15. 15

    Wang, H.H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 16

    Baggett, N.E., Zhang, Y. & Gross, C.A. Global analysis of translation termination in E. coli. PLoS Genet. 13, e1006676 (2017).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  17. 17

    Mora, L., Heurgué-Hamard, V., de Zamaroczy, M., Kervestin, S. & Buckingham, R.H. Methylation of bacterial release factors RF1 and RF2 is required for normal translation termination in vivo. J. Biol. Chem. 282, 35638–35645 (2007).

    CAS  PubMed  Article  Google Scholar 

  18. 18

    Lacourciere, G.M., Levine, R.L. & Stadtman, T.C. Direct detection of potential selenium delivery proteins by using an Escherichia coli strain unable to incorporate selenium from selenite into proteins. Proc. Natl. Acad. Sci. USA 99, 9150–9153 (2002).

    CAS  PubMed  Article  Google Scholar 

  19. 19

    Scheer, H. & Zhao, K.H. Biliprotein maturation: the chromophore attachment. Mol. Microbiol. 68, 263–276 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20

    Rodriguez, E.A. et al. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein. Nat. Methods 13, 763–769 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. 21

    Miklos, A.E. et al. Structure-based design of supercharged, highly thermoresistant antibodies. Chem. Biol. 19, 449–455 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22

    Cheng, Q. & Arnér, E.S.J. Selenocysteine insertion at a predefined UAG codon in a Release Factor 1 (RF1)-depleted Escherichia coli host strain bypasses species barriers in recombinant selenoprotein translation. J. Biol. Chem. 292, 5476–5487 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23

    Fan, Z., Song, J., Guan, T., Lv, X. & Wei, J. Efficient expression of glutathione peroxidase with chimeric tRNA in Amber-less Escherichia coli. ACS Synth. Biol. 7, 249–257 (2018).

    CAS  PubMed  Article  Google Scholar 

  24. 24

    Walewska, A. et al. Integrated oxidative folding of cysteine/selenocysteine containing peptides: improving chemical synthesis of conotoxins. Angew. Chem. Int. Edn Engl. 48, 2221–2224 (2009).

    CAS  Article  Google Scholar 

  25. 25

    Gaciarz, A. et al. Systematic screening of soluble expression of antibody fragments in the cytoplasm of E. coli. Microb. Cell Fact. 15, 22 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  26. 26

    Lobstein, J. et al. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microb. Cell Fact. 11, 753 (2012).

    Article  CAS  Google Scholar 

  27. 27

    Zhang, Y., Cui, W., Zhang, H., Dewald, H.D. & Chen, H. Electrochemistry-assisted top-down characterization of disulfide-containing proteins. Anal. Chem. 84, 3838–3842 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. 28

    Zhao, D.S., Gregorich, Z.R. & Ge, Y. High throughput screening of disulfide-containing proteins in a complex mixture. Proteomics 13, 3256–3260 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. 29

    Kraj, A., Brouwer, H.J., Reinhoud, N. & Chervet, J.P. A novel electrochemical method for efficient reduction of disulfide bonds in peptides and proteins prior to MS detection. Anal. Bioanal. Chem. 405, 9311–9320 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30

    Valentine, S.J., Anderson, J.G., Ellington, A.D. & Clemmer, D.E. Disulfide-intact and -reduced lysozyme in the gas phase:conformations and pathways of folding and unfolding. J. Phys. Chem. B 101, 3891–3900 (1997).

    CAS  Article  Google Scholar 

  31. 31

    Muttenthaler, M. et al. Modulating oxytocin activity and plasma stability by disulfide bond engineering. J. Med. Chem. 53, 8585–8596 (2010).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  32. 32

    Armishaw, C.J. et al. Alpha-selenoconotoxins, a new class of potent alpha7 neuronal nicotinic receptor antagonists. J. Biol. Chem. 281, 14136–14143 (2006).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  33. 33

    Pelat, T. et al. Isolation of a human-like antibody fragment (scFv) that neutralizes ricin biological activity. BMC Biotechnol. 9, 60 (2009).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  34. 34

    Kuznetsov, G. et al. Optimizing complex phenotypes through model-guided multiplex genome engineering. Genome Biol. 18, 100 (2017).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  35. 35

    Amiram, M. et al. Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids. Nat. Biotechnol. 33, 1272–1279 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 36

    Rovner, A.J. et al. Recoded organisms engineered to depend on synthetic amino acids. Nature 518, 89–93 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. 37

    Mandell, D.J. et al. Biocontainment of genetically modified organisms by synthetic protein design. Nature 518, 55–60 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38

    Tack, D.S. et al. Addicting diverse bacteria to a noncanonical amino acid. Nat. Chem. Biol. 12, 138–140 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  39. 39

    Wannier, T.M. et al. Long-term adaptive evolution of genomically recoded. Escherichia coli. Preprint @ https://www.biorxiv.org/content/early/2017/07/12/162834 (2017).

  40. 40

    Arai, K. et al. Preparation of selenoinsulin as a long-lasting insulin analogue. Angew. Chem. Int. Edn Engl. 56, 5522–5526 (2017).

    CAS  Article  Google Scholar 

  41. 41

    Metanis, N. & Hilvert, D. Strategic use of non-native diselenide bridges to steer oxidative protein folding. Angew. Chem. Int. Edn Engl. 51, 5585–5588 (2012).

    CAS  Article  Google Scholar 

  42. 42

    Onderko, E.L., Silakov, A., Yosca, T.H. & Green, M.T. Characterization of a selenocysteine-ligated P450 compound I reveals direct link between electron donation and reactivity. Nat. Chem. 9, 623–628 (2017).

    CAS  PubMed  Article  Google Scholar 

  43. 43

    Vandemeulebroucke, A., Aldag, C., Stiebritz, M.T., Reiher, M. & Hilvert, D. Kinetic consequences of introducing a proximal selenocysteine ligand into cytochrome P450cam. Biochemistry 54, 6692–6703 (2015).

    CAS  PubMed  Article  Google Scholar 

  44. 44

    Hondal, R.J. Using chemical approaches to study selenoproteins-focus on thioredoxin reductases. Biochim. Biophys. Acta 1790, 1501–1512 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. 45

    Yu, Y. et al. Characterization and structural analysis of human selenium-dependent glutathione peroxidase 4 mutant expressed in Escherichia coli. Free Radic. Biol. Med. 71, 332–338 (2014).

    CAS  PubMed  Article  Google Scholar 

  46. 46

    Mannes, A.M., Seiler, A., Bosello, V., Maiorino, M. & Conrad, M. Cysteine mutant of mammalian GPx4 rescues cell death induced by disruption of the wild-type selenoenzyme. FASEB J. 25, 2135–2144 (2011).

    CAS  PubMed  Article  Google Scholar 

  47. 47

    Dery, L. et al. Accessing human selenoproteins through chemical protein synthesis. Chem. Sci. (Camb.) 8, 1922–1926 (2017).

    CAS  Article  Google Scholar 

  48. 48

    Cohen, D.T., Zhang, C., Pentelute, B.L. & Buchwald, S.L. An umpolung approach for the chemoselective arylation of selenocysteine in unprotected peptides. J. Am. Chem. Soc. 137, 9784–9787 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. 49

    Byrom, M., Bhadra, S., Jiang, Y.S. & Ellington, A.D. Exquisite allele discrimination by toehold hairpin primers. Nucleic Acids Res. 42, e120 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  50. 50

    Shaw, J.B. et al. Complete protein characterization using top-down mass spectrometry and ultraviolet photodissociation. J. Am. Chem. Soc. 135, 12646–12651 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

Funding from the Welch Foundation (F-1654 to A.D.E. and F-1155 to J.S.B.), the NSSEFF (FA9550-10-1-0169 to A.D.E.), the NSF (CHE-1402753 to J.S.B.), the ARO (SP0036191-PROJ0009952), and the NIH NCI (5K99CA207870-02 to R.T.) is acknowledged.

Author information

Affiliations

Authors

Contributions

R.T. designed the experiments and performed the strain evolution, protein purification, and protein characterization. Bioinformatic analysis was done by R.S. MAGE was conducted by R.T. and R.S. The fluorescent reporter was developed by R.T. and S.d′O. Mass spectrometry was performed by D.R.K., V.C.C., and J.S.B. qPCR was performed by M.B. The manuscript was written by R.T. with support from A.D.E. R.T. and A.D.E. supervised all aspects of the study.

Corresponding authors

Correspondence to Ross Thyer or Andrew D Ellington.

Ethics declarations

Competing interests

R.T. and A.D.E. have equity in GRO Biosciences, a company developing protein therapeutics, and receive royalties from licensing material described in this manuscript.

Integrated supplementary information

Supplementary Figure 1 Selenocysteine biosynthesis is toxic and incorporation is poorly maintained.

Dilution plating of RTΔA cells transformed with pRSF-UX-SelA (wild-type or truncated) and p15A-NMC-A (wild-type or C69U-C238U) demonstrates two related phenomena; toxicity of selenocysteine biosynthesis and loss of selenocysteine incorporation. In addition to expressing the NMC-A β-lactamase, p15A-NMC-A confers tetracycline resistance. (a-b) The presence of functional SelA (panel a) drastically decreases cell growth and viability following transformation compared to cells which lack SelA (panel b). Carbenicillin resistance is directly correlated with tetracycline resistance. (c-d) Cells transformed with NMC-A C69U-C238U and functional SelA can grow in the presence of carbenicillin (panel c), while those with a truncated version of SelA do not (panel d). However, carbenicillin resistant cells are 10-fold less abundant than tetracycline resistant cells (panel c) indicating not all transformants are capable of incorporating selenocysteine (at levels sufficient to support β-lactamase activity). Plates are representative from two biologically independent series.

Supplementary Figure 2 Growth curves in rich media (LB) of parental strains and descendant populations evolved under β-lactam stress.

The labels indicate the carbenicillin concentration and the curves are plotted with a line showing the mean and shading representing ± S.E.M. where n = three independent biological replicates. (a) Δ parental strain and evolved populations β_Δ1-3. (b) CC parental strain and evolved populations β_CC1-3. (c) UC parental strain and evolved populations β_UC1-3. (d) UU parental strain and evolved populations β_UU1-3. Evolved populations have increased growth rate at given carbenicillin concentrations, reach higher culture density and show greater resistance to carbenicillin compared to their respective parental strains.

Supplementary Figure 3 Growth curves in selenium free media (MOPS EZ) of parental strains and descendant populations evolved under β-lactam stress.

The labels indicate the media additives and the curves are plotted with a line showing the mean and shading representing ± S.E.M. where n = three independent biological replicates. Carbenicillin and Na2SeO3 were added at concentrations of 100 μg.mL and 1 μM respectively. (a) Δ parental strain and evolved populations β_Δ1-3. (b) CC parental strain and evolved populations β_CC1-3. (c) UC parental strain and evolved populations β_UC1-3. (d) UU parental strain and evolved populations β_UU1-3. The Δ and UU parental strains had severely impaired growth in MOPS EZ media, particularly when grown in 96-well plates. Evolved populations have increased growth rate and reach higher culture density compared to parental strains. All β_UC and β_UU populations retained selenocysteine dependence, requiring selenite supplementation to grow in the presence of carbenicillin.

Supplementary Figure 4 Growth curves in rich media (LB) at 37 °C of parental strains compared to descendant populations evolved under thermal stress.

The curves are plotted with a line showing the mean and shading representing ± S.E.M. where n = three independent biological replicates. (a) Δ parental strain compared to evolved populations T_Δ1-3. (b) CC parental strain compared to evolved populations T_CC1-3. (c) UC parental strain compared to evolved populations T_UC1-3. (d) UU parental strain compared to evolved populations T_UU1-3. Evolved populations generally had poorer growth rates and reached lower culture density compared to parental strains.

Supplementary Figure 5 Growth curves in selenium free media (MOPS EZ) of parental strains and descendant populations evolved under thermal stress.

The labels indicate the media additives and the curves are plotted with a line showing the mean and shading representing ± S.E.M. where n = three independent biological replicates. Carbenicillin and Na2SeO3 were added at concentrations of 100 μg.mL and 1 μM respectively. (a) Δ parental strain and evolved populations T_Δ1-3. (b) CC parental strain and evolved populations T_CC1-3. (c) UC parental strain and evolved populations T_UC1-3. (d) UU parental strain and evolved populations T_UU1-3. The Δ and UU parental strains had severely impaired growth in MOPS EZ media, particularly when grown in 96-well plates. Evolved populations have increased growth rate and reach higher culture density compared to parental strains. All T_UC and T_UU populations retained selenocysteine dependence, requiring selenite supplementation to grow in the presence of carbenicillin.

Supplementary Figure 6 Mass spectrometry of GFP X177 expressed in β_Δ1 and T_Δ1 cells reveals that UAG codons are read as glutamine by near cognate suppression.

(a) Mass spectrum of intact GFP X177 expressed in β_Δ1 cells. (b) Deconvoluted mass spectrum of GFP X177 expressed in β_Δ1 cells reveals two species resulting from partial loss of the N-terminal methionine residue. (c) Experimentally determined monoisotopic masses (27928.91 Da and 27797.87 Da) match the theoretical masses of (27928.85 Da and 27797.81 Da) of GFP containing a glutamine residue at position 177. (d) Mass spectrum of intact GFP X177 expressed in T_Δ1 cells. (e) Deconvoluted mass spectrum of GFP X177 expressed in T_Δ1 cells. (f) Experimentally determined monoisotopic masses (27928.91 Da and 27797.88 Da) match the theoretical masses of (27928.85 Da and 27797.81 Da) of GFP containing a glutamine residue at position 177. All spectra are representative from a minimum of two technical replicates.

Supplementary Figure 7 Growth curves of RTΔA cells containing point mutants in oxidative stress and selenite resistance.

Growth curves of RTΔA cells containing point mutants in oxidative stress and selenite resistance genes in rich and defined media ± 1 μM Na2SeO3. The curves are plotted with a line showing the mean and shading representing ± S.E.M. where n = three independent biological replicates. (a) Growth curves of RTΔA containing a constitutively active variant of OxyR (A233T) observed during evolution. The A233T mutation does not provide any clear benefits to cell fitness compared to a wild-type control. (b-d) Growth curves of RTΔA containing T69I, T73A or H153Y mutations respectively in CysK, which are expected to strongly inhibit cysteine biosynthesis, compared to a wild-type control. Mutations are not observed to have a significant impact on cell growth.

Supplementary Figure 8 Mutant cysK allele retention and plasmid copy number determination.

Analysis of mutant cysK allele frequency by qPCR in cell populations passaged for 125 generations in the presence of 10 μM Na2SeO3. At generation 0 (purple bars), cell populations contained 50% wild-type cysK and 50% mutant cysK. Bars represent the mean ± S.E.M. (a) Detection of wild-type cysK (T69) in WT:T69I populations ± Na2SeO3. Wild-type cysK is detected at a similar frequency in both the presence and absence of Na2SeO3. (b) In contrast, mutant T69I undetectable (Cq = ~ 35) after 125 generations in all populations. (c) Detection of wild-type cysK (T73) in WT:T73A populations does not change ± Na2SeO3. (d) The cysK T73A mutant is lost from the populations when cultured in the absence of Na2SeO3 (light blue). In contrast, in the presence of Na2SeO3 (dark blue) the mutant allele is retained. (e) Detection of wild-type cysK (H153) in WT:H153Y populations is not affected by Na2SeO3 treatment. (f) Similar to T73A, the H135Y mutant is undetectable after serial passage in media without selenite (light blue), but is partially retained when selenite is present (dark blue). Abundance of pCDF (g) and pRSF (h) plasmids in RTΔA cells with mutations in poly(A) polymerase I (pcnB) and DNA polymerase I (polA). All mutations significantly decreased plasmid abundance compared to wild-type cells. Bars represent the mean ± S.E.M.

Supplementary Figure 9 Growth curves of C321.ΔA and prfB (release factor 2) single point mutants in RTΔA cells in rich and defined media.

The curves are plotted with a line showing the mean and shading representing ± S.E.M. where n = three independent biological replicates. (a) Growth of RTΔA ftsA G124E shows minor improvement in defined media. (b) Growth of RTΔA hemA P196L shows significant improvement in defined media. (c) No improvement was observed in either rich or defined media for RTΔA pta P673L. (d) Growth of RTΔA yeeJ S1467P shows significant improvement in defined media. (e) The T246A mutation in release factor two significantly improves growth of RTΔA cells in defined media. (f) No improvement was observed in either rich or defined media for RTΔA prfB N276D. (g) A T246A-N276D double mutant was generated to investigate potential synergy between T246A and N276D which co-occurred in one evolved population. Observed growth improvement is due to T246A mutation only. (h) Growth of RTΔA prfB K282R shows minor improvement in defined media.

Supplementary Figure 10 Mass spectrometry of GFP X177 expressed in β_CC1 and T_CC1 cells in the absence of Na2SeO3 confirms the incorporation of serine.

(a) Mass spectrum of intact GFP X177 expressed in β_CC1 cells. (b) Deconvoluted mass spectrum of GFP X177 expressed in β_CC1 cells reveals two species resulting from partial loss of the N-terminal methionine residue. (c) Experimentally determined monoisotopic masses (27887.87 Da and 27756.84 Da) match the theoretical masses of (27887.83 Da and 27756.79 Da) of GFP containing a serine residue at position 177. (d) Mass spectrum of intact GFP X177 expressed in T_CC1 cells. (e) Deconvoluted mass spectrum of GFP X177 expressed in T_CC1 cells. (f) Experimentally determined monoisotopic masses (27887.85 Da and 27756.84 Da) match the theoretical masses of (27887.83 Da and 27756.79 Da) of GFP containing a serine residue at position 177. All spectra are representative from a minimum of two technical replicates.

Supplementary Figure 11 Fluorescence screen for retention of selenocysteine incorporation in evolved populations using seleno-smURFP.

Internal controls are located below the streaks are (from left to right) ΔsmURFP, smURFP-C52, seleno-smURFP (U52) and smURFP-S52. (a) β_Δ populations deficient in selenocysteine incorporation were non-fluorescent. Both the non-selenocysteine dependent population (β_CC) and the two selenocysteine dependent populations (β_UC and β_UU) had almost complete retention of the trait. (b) T_Δ populations deficient in selenocysteine incorporation were non-fluorescent. The selenocysteine dependent T_UC and T_UU populations had almost complete retention of the trait, although no transformants could be obtained from T_UC3 and only a small number from T_UU3. In contrast, retention was highly variable across the three T_CC populations which were not selenocysteine dependent. Note, in this composite image the T_UU2 plate was imaged after an additional 24 hours of incubation at 37 °C due to very slow growth of the streaked cells using an exposure time of 0.5 seconds. The internal controls are consistent with the other plates and clear differences are observed between the streaked cells and the non-fluorescent smURFP-S52 control.

Supplementary Figure 12 Mass spectrometry of two recombinant selenoproteins expressed in β_UU3 cells confirms highly efficient selenocysteine incorporation and correct formation of a diselenide bond.

(a) Mass spectrum of intact EcDHFR U39-U85. (b) Deconvoluted mass spectrum of EcDHFR U39-U85. (c) Experimentally determined monoisotopic mass (19054.13 Da) matches the theoretical mass (19054.14 Da) of EcDHFR U39-U85 containing a diselenide bond. The experimental mass has been adjusted to account for deconvolution error arising from the unusual isotope distribution of selenium. (d) UVPD mass spectrum of the 17+ charge state. (e) UVPD fragment map shows a lack of detectable fragments between U39 and U85 which confirms diselenide bond formation. (f) Mass spectrum of intact GFP U135-U177. (g) Deconvoluted mass spectrum of GFP U135-U177. (h) Experimentally determined monoisotopic masses (28013.64 Da and 27882.61 Da) match the theoretical masses of (28013.66 Da and 278861 Da) of GFP U135-U177 containing a diselenide bond ± the N-terminal methionine residue. The experimental masses have been adjusted to account for deconvolution error. (i) UVPD mass spectrum of the 28+ charge state. (j) UVPD fragment map shows a lack of detectable fragments between U135 and U177 which confirms diselenide bond formation. The lack of fragments near Y66 corresponds to the mature GFP chromophore. All spectra are representative from a minimum of two technical replicates.

Supplementary Figure 13 Mass spectrometry of two recombinant selenoproteins expressed in β_UU3 cells confirms diselenide bonds can replace essential structural disulfide bonds in therapeutically relevant protein families.

(a) Mass spectrum of intact seleno-anti-MS2 scFv. (b) Deconvoluted mass spectrum of seleno-anti-MS2 scFv. (c) Experimentally determined monoisotopic mass (31855.08 Da) matches the theoretical mass (31855.06 Da) of seleno-anti-MS2 containing a two diselenide bonds. The experimental mass has been adjusted to account for deconvolution error arising from the unusual isotope distribution of selenium. (d) UVPD mass spectrum of the 24+ charge state. (e) UVPD fragment map shows a lack of detectable fragments between U42 and U116 in the VH domain, and U179 and U249 in the VL domain, which confirms correct formation of both diselenide bonds. (f) Mass spectrum of intact seleno-hGH. (g) Deconvoluted mass spectrum of seleno-hGH. (h) Experimentally determined monoisotopic mass (23400.27 Da) matches the theoretical mass (23499.27 Da) of seleno-hGH containing two diselenide bonds. The experimental mass has been adjusted to account for deconvolution error. (i) UVPD mass spectrum of the 16+ charge state. (j) UVPD fragment map shows a lack of detectable fragments between U54 and U166 and U183 and U190 which confirms correct formation of both diselenide bonds. All spectra are representative from a minimum of two technical replicates.

Supplementary Figure 14 Mass spectra of Herceptin (Trastuzumab) scFvs.

(a) Mass spectrum of intact wild-type Herceptin-scFv expressed under reducing conditions. (b) Deconvoluted mass spectrum of wild-type Herceptin-scFv. (c) Experimentally determined monoisotopic mass (27172.05 Da) matches the theoretical mass (27172.08 Da) of wild-type Herceptin scFv containing reduced cysteine residues. (d) UVPD mass spectrum of the 29+ charge state of wild-type Herceptin. (e) UVPD fragment map shows extensive fragmentation across both the VL domain, and the VH domain, which confirms a lack of disulfide bonds. (f) Mass spectrum of intact wild-type Herceptin-scFv expressed under oxidizing conditions. (g) Deconvoluted mass spectrum of wild-type Herceptin scFv. (h) Experimentally determined monoisotopic mass (27170.12 Da) matches the theoretical mass (27170.07 Da) of wild-type Herceptin-scFv containing a single disulfide bond. (i) UVPD mass spectrum of the 23+ charge state of wild-type Herceptin-scFv. (j) UVPD fragment map shows extensive fragmentation across the VL domain, but a large fragment gap between C149 and C223 in the VH domain, which confirms the formation of one disulfide bond. (k) Mass spectrum of intact seleno-Herceptin-scFv expressed in β_UU3 cells. (l) Deconvoluted mass spectrum of seleno-Herceptin-scFv. (m) Experimentally determined monoisotopic mass (27359.88 Da) matches the theoretical mass (27359.83 Da) of seleno-Herceptin-scFv containing two diselenide bonds. The experimental mass has been adjusted to account for deconvolution error arising from the unusual isotope distribution of selenium. (n) UVPD mass spectrum of the 20+ charge state of seleno-Herceptin-scFv. (o) The UVPD fragment map shows a lack of detectable fragments between U24 and U89 in the VL domain, and U149 and U223 in the VH domain, which confirms correct formation of both diselenide bonds. All spectra are representative from a minimum of two technical replicates.

Supplementary Figure 15 Mass spectra of anti-RCA scFvs.

(a) Mass spectrum of intact wild-type anti-RCA scFv. (b) Deconvoluted mass spectrum of wild-type anti-RCA scFv. (c) Experimentally determined monoisotopic mass (27002.21 Da) matches the theoretical mass (27002.17 Da) of wild-type anti-RCA scFv containing a two disulfide bonds. (d) UVPD mass spectrum of the 20+ charge state. (e) The UVPD fragment map shows a lack of detectable fragments between C23 and C97 in the VH domain, and C159 and C224 in the VL domain, which confirms correct formation of both disulfide bonds. (f) Mass spectrum of intact seleno-anti-RCA. (g) Deconvoluted mass spectrum of seleno-anti-RCA. (h) Experimentally determined monoisotopic mass (27193.96 Da) matches the theoretical mass (27193.94 Da) of seleno-anti-RCA containing two diselenide bonds. The experimental mass has been adjusted to account for deconvolution error arising from the unusual isotope distribution of selenium. (i) UVPD mass spectrum of the 20+ charge state. (j) The UVPD fragment map shows a lack of detectable fragments between U23 and U97 in the VH domain, and U159 and U224 in the VL domain, which confirms correct formation of both diselenide bonds. All spectra are representative from a minimum of two technical replicates.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15 (PDF 2978 kb)

Life Sciences Reporting Summary (PDF 141 kb)

Supplementary Table 1

Genes containing in-frame TAG codons which enriched in evolved populations. (XLSX 15 kb)

Supplementary Table 2

SNPs enriched in populations evolved under β-lactam stress. (XLSX 10 kb)

Supplementary Table 3

SNPs enriched in populations evolved under thermal stress. (XLSX 11 kb)

Supplementary Tables

Supplementary Tables 4–13 (PDF 281 kb)

Supplementary Note 1

Supplementary Note 1 (PDF 341 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Thyer, R., Shroff, R., Klein, D. et al. Custom selenoprotein production enabled by laboratory evolution of recoded bacterial strains. Nat Biotechnol 36, 624–631 (2018). https://doi.org/10.1038/nbt.4154

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing