Article | Published:

Custom selenoprotein production enabled by laboratory evolution of recoded bacterial strains

Nature Biotechnology volume 36, pages 624631 (2018) | Download Citation

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.

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NCBI Reference Sequence

Referenced accessions

Protein Data Bank

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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

  1. Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas, USA.

    • Ross Thyer
    • , Raghav Shroff
    • , Simon d'Oelsnitz
    • , Michelle Byrom
    •  & Andrew D Ellington
  2. Department of Chemistry, University of Texas at Austin, Austin, Texas, USA.

    • Dustin R Klein
    • , Victoria C Cotham
    •  & Jennifer S Brodbelt

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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.

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.

Corresponding authors

Correspondence to Ross Thyer or Andrew D Ellington.

Integrated supplementary information

Supplementary figures

  1. 1.

    Selenocysteine biosynthesis is toxic and incorporation is poorly maintained.

  2. 2.

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

  3. 3.

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

  4. 4.

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

  5. 5.

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

  6. 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.

  7. 7.

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

  8. 8.

    Mutant cysK allele retention and plasmid copy number determination.

  9. 9.

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

  10. 10.

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

  11. 11.

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

  12. 12.

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

  13. 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.

  14. 14.

    Mass spectra of Herceptin (Trastuzumab) scFvs.

  15. 15.

    Mass spectra of anti-RCA scFvs.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Tables

    Supplementary Tables 4–13

  4. 4.

    Supplementary Note 1

    Supplementary Note 1

Excel files

  1. 1.

    Supplementary Table 1

    Genes containing in-frame TAG codons which enriched in evolved populations.

  2. 2.

    Supplementary Table 2

    SNPs enriched in populations evolved under β-lactam stress.

  3. 3.

    Supplementary Table 3

    SNPs enriched in populations evolved under thermal stress.

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DOI

https://doi.org/10.1038/nbt.4154