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Directed evolution improves the catalytic efficiency of TEV protease

A Publisher Correction to this article was published on 06 January 2020

This article has been updated


Tobacco etch virus protease (TEV) is one of the most widely used proteases in biotechnology because of its exquisite sequence specificity. A limitation, however, is its slow catalytic rate. We developed a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is read out via proteolytic release of a membrane-anchored transcription factor, and we temporally regulate access to TEV’s cleavage substrate using a photosensory LOV domain. By gradually decreasing light exposure time, we enriched faster variants of TEV over multiple rounds of selection. Our TEV-S153N mutant (uTEV1Δ), when incorporated into the calcium integrator FLARE, improved the signal/background ratio by 27-fold, and enabled recording of neuronal activity in culture with 60-s temporal resolution. Given the widespread use of TEV in biotechnology, both our evolved TEV mutants and the directed-evolution platform used to generate them could be beneficial across a wide range of applications.

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Fig. 1: Yeast platform for directed evolution of high-turnover, low-affinity proteases.
Fig. 2: Characterization of evolved low-affinity proteases (uTEV1Δ and uTEV2Δ) in yeast and in vitro.
Fig. 3: Yeast platform applied to the evolution of high-affinity proteases.
Fig. 4: Characterization of evolved low-affinity TEVΔ proteases in mammalian cells and incorporation into FLARE and SPARK tools.

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Additional data beyond that provided in the Figures and Supplementary Information are available from the corresponding author upon request.

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  1. Liu, Q. et al. A photoactivatable botulinum neurotoxin for inducible control of neurotransmission. Neuron 101, 863–875 (2019).

  2. Smart, A. D. et al. Engineering a light-activated caspase-3 for precise ablation of neurons in vivo. Proc. Natl Acad. Sci. USA 114, E8174–E8183 (2017).

    CAS  Article  Google Scholar 

  3. Lin, M. Z., Glenn, J. S. & Tsien, R. Y. A drug-controllable tag for visualizing newly synthesized proteins in cells and whole animals. Proc. Natl Acad. Sci. USA 105, 7744–7749 (2008).

    CAS  Article  Google Scholar 

  4. Schuster, B. S. et al. Controllable protein phase separation and modular recruitment to form responsive membraneless organelles. Nat. Commun. 9, 2985 (2018).

    Article  Google Scholar 

  5. Gao, X. J., Chong, L. S., Kim, M. S. & Elowitz, M. B. Programmable protein circuits in living cells. Science 361, 1252–1258 (2018).

    CAS  Article  Google Scholar 

  6. Fink, T. et al. Design of fast proteolysis-based signaling and logic circuits in mammalian cells. Nat. Chem. Biol. 15, 115–122 (2019).

    CAS  Article  Google Scholar 

  7. Copeland, M. F., Politz, M. C., Johnson, C. B., Markley, A. L. & Pfleger, B. F. A transcription activator–like effector (TALE) induction system mediated by proteolysis. Nat. Chem. Biol. 12, 254–260 (2016).

    CAS  Article  Google Scholar 

  8. Wang, W. et al. A light- and calcium-gated transcription factor for imaging and manipulating activated neurons. Nat. Biotechnol. 35, 864–871 (2017).

    CAS  Article  Google Scholar 

  9. Lee, D., Hyun, J. H., Jung, K., Hannan, P. & Kwon, H.-B. A calcium- and light-gated switch to induce gene expression in activated neurons. Nat. Biotechnol. 35, 858–863 (2017).

    CAS  Article  Google Scholar 

  10. Barnea, G. et al. The genetic design of signaling cascades to record receptor activation. Proc. Natl Acad. Sci. USA 105, 64–69 (2008).

    CAS  Article  Google Scholar 

  11. Kim, M. W. et al. Time-gated detection of protein-protein interactions with transcriptional readout. eLife 6, e30233 (2017).

    Article  Google Scholar 

  12. Kim, C. K., Cho, K. F., Kim, M. W. & Ting, A. Y. Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions. eLife 8, e43826 (2019).

    Article  Google Scholar 

  13. Parks, T. D., Howard, E. D., Wolpert, T. J., Arp, D. J. & Dougherty, W. G. Expression and purification of a recombinant tobacco etch virus NIa proteinase: biochemical analyses of the full-length and a naturally occurring truncated proteinase form. Virology 210, 194–201 (1995).

    CAS  Article  Google Scholar 

  14. Evnin, L. B., Vásquez, J. R. & Craik, C. S. Substrate specificity of trypsin investigated by using a genetic selection. Proc. Natl Acad. Sci. USA 87, 6659–6663 (1990).

    CAS  Article  Google Scholar 

  15. Estell, D. A., Graycar, T. P. & Wells, J. A. Engineering an enzyme by site-directed mutagenesis to be resistant to chemical oxidation. J. Biol. Chem. 260, 6518–6521 (1985).

    CAS  Article  Google Scholar 

  16. Packer, M. S., Rees, H. A. & Liu, D. R. Phage-assisted continuous evolution of proteases with altered substrate specificity. Nat. Commun. 8, 956 (2017).

    Article  Google Scholar 

  17. Yi, L. et al. Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries. Proc. Natl Acad. Sci. USA 110, 7229–7234 (2013).

    CAS  Article  Google Scholar 

  18. Lam, S. S. et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 12, 51–54 (2015).

    CAS  Article  Google Scholar 

  19. Branon, T. C. et al. Efficient proximity labeling in living cells and organisms with TurboID. Nat. Biotechnol. 36, 880–887 (2018).

    CAS  Article  Google Scholar 

  20. Martell, J. D. et al. A split horseradish peroxidase for the detection of intercellular protein–protein interactions and sensitive visualization of synapses. Nat. Biotechnol. 34, 774–780 (2016).

    CAS  Article  Google Scholar 

  21. Han, Y. et al. Directed evolution of split APEX2 peroxidase. ACS Chem. Biol. 14, 619–635 (2019).

  22. Kennedy, M. J. et al. Rapid blue-light-mediated induction of protein interactions in living cells. Nat. Methods 7, 973–975 (2010).

    CAS  Article  Google Scholar 

  23. Kapust, R. B. et al. Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng. 14, 993–1000 (2001).

    CAS  Article  Google Scholar 

  24. Phan, J. et al. Structural basis for the substrate specificity of tobacco etch virus protease. J. Biol. Chem. 277, 50564–50572 (2002).

    CAS  Article  Google Scholar 

  25. Raran-Kurussi, S., Tözsér, J., Cherry, S., Tropea, J. E. & Waugh, D. S. Differential temperature dependence of tobacco etch virus and rhinovirus 3C protease. s. Anal. Biochem. 436, 142–144 (2013).

    CAS  Article  Google Scholar 

  26. Kostallas, G., Löfdahl, P.-Å. & Samuelson, P. Substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay. PLoS One 6, e16136 (2011).

    CAS  Article  Google Scholar 

  27. Li, Q. et al. Profiling protease specificity: combining yeast ER sequestration screening (YESS) with next generation sequencing. ACS Chem. Biol. 12, 510–518 (2017).

    CAS  Article  Google Scholar 

  28. Kapust, R. B., Tözsér, J., Copeland, T. D. & Waugh, D. S. The P1′ specificity of tobacco etch virus protease. Biochem. Biophys. Res. Commun. 294, 949–955 (2002).

    CAS  Article  Google Scholar 

  29. Thomsen, M. C. F. & Nielsen, M. Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion. Nucleic Acids Res 40, W281–W287 (2012).

    CAS  Article  Google Scholar 

  30. Cabrita, L. D. et al. Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci. 16, 2360–2367 (2007).

    CAS  Article  Google Scholar 

  31. Sente, A. et al. Molecular mechanism of modulating arrestin conformation by GPCR phosphorylation. Nat. Struct. Mol. Biol. 25, 538–545 (2018).

    CAS  Article  Google Scholar 

  32. Martell, J. D. et al. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat. Biotechnol. 30, 1143–1148 (2012).

    CAS  Article  Google Scholar 

  33. Strickland, D. et al. Rationally improving LOV domain–based photoswitches. Nat. Methods 7, 623–626 (2010).

    CAS  Article  Google Scholar 

  34. Turk, B. E., Huang, L. L., Piro, E. T. & Cantley, L. C. Determination of protease cleavage site motifs using mixture-based oriented peptide libraries. Nat. Biotechnol. 19, 661–667 (2001).

    CAS  Article  Google Scholar 

  35. Wiita, A. P., Seaman, J. E. & Wells, J. A. Global analysis of cellular proteolysis by selective enzymatic labeling of protein N-termini. Methods Enzymol. 544, 327–358 (2014).

    CAS  Article  Google Scholar 

  36. Kim, J. H. et al. High cleavage efficiency of a 2 A peptide derived from porcine Teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6, e18556 (2011).

    CAS  Article  Google Scholar 

  37. Ottoz, D. S. M., Rudolf, F. & Stelling, J. Inducible, tightly regulated and growth condition-independent transcription factor in saccharomyces cerevisiae. Nucleic Acids Res. 42, e130–e130 (2014).

    Article  Google Scholar 

  38. Peng, B., Williams, T. C., Henry, M., Nielsen, L. K. & Vickers, C. E. Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: a comparison of yeast promoter activities. Microb. Cell Fact. 14, 91 (2015).

    Article  Google Scholar 

  39. Swiech, L. et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat. Biotechnol. 33, 102–106 (2015).

    CAS  Article  Google Scholar 

  40. Loh, K. H. et al. Proteomic analysis of unbounded cellular compartments: synaptic clefts. Cell 166, 1295–1307.e21 (2016).

    CAS  Article  Google Scholar 

  41. Tropea, J. E., Cherry, S. & Waugh, D. S. Expression and purification of soluble His6-tagged TEV protease. Methods Mol. Biol. 498, 297–307 (2009).

    CAS  Article  Google Scholar 

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We are grateful to Stanford, the Chan Zuckerberg Biohub, the Beckman Technology Development Seed Grant and NIH (R01 MH119353) for support of this work. FACS was performed at the MIT Koch Institute Flow Cytometry Core and at the Stanford Shared FACS Facility. W. Wang (University of Michigan) provided plasmids and advice. L. Ning (Stanford University) provided rat brain tissue. A. G. Johnson (Stanford) gave advice on TEV expression, and N. Samiylenko helped reproduce some experiments. B. Babin, J. Yim and M. Bogyo (Stanford University) provided access to their HPLC. G. Liu (MIT) built the LED box used for blue light irradiation of cells. M. Djuristic (Stanford University) assisted with electrical stimulation of neurons. M.I.S. was supported by an EMBO long-term post-doctoral fellowship (ALTF 1022-2015).

Author information

Authors and Affiliations



M.I.S. performed all the experiments. M.I.S. and A.Y.T. designed the research, analyzed the data, wrote and edited the paper.

Corresponding author

Correspondence to Alice Y. Ting.

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Competing interests

A.Y.T. and M.I.S. have filed a patent application covering some aspects of this work (US provisional application 62/906,373; CZB file CZB-123S-P1; Stanford file S19-269; KT file 103182-1132922-002400PR).

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Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Optimization of membrane-anchored transcription factor for yeast directed evolution.

Related to Fig. 1c. a, BY4741 yeast constitutively expressing STE2-citrine or STE2Δ(1-300)-citrine; the latter have much improved surface localization. Scale bars, 10 μm. b, Left four columns, FACS plots showing yeast cells 6 h after 45-min blue-light irradiation. Percentages reflect fraction of cells with Citrine signal (cells that release TF to drive Citrine expression) and are given in the table in Fig. 1c. Right four columns, Control cells without light exposure. Each condition performed twice; n = 10,000 cells. c, Optimization of the LexA transcriptional activator. Yeast co-expressing the TF and mCherry-CRY-TEVΔ were analyzed by FACS at 6 h after variable amounts of blue-light exposure. Percentages reflect the fraction of Citrine-positive cells. d, Optimizing the time for reporter transcription and translation. FACS plots collected at various timepoints after 45-min blue-light exposure to induce TF release. Percentages are the fraction of cells in Q1 and Q2 FACS quadrants. Each plot is representative of two replicates; n = 20,000 cells. We selected 6 h as our expression time window.

Supplementary Figure 2 Analysis of selected yeast populations and TEV clones.

Related to Fig. 1f. a, Same as Fig. 1f, but more conditions are shown. B, Sequencing after round 3. 24 clones were sequenced and mutations were found in each, relative to original TEVΔ, are shown.

Supplementary Figure 3 Characterization of evolved TEVΔ mutants in yeast.

Related to Fig. 2a. a, FACS plots were collected 6 h after blue-light exposure for the indicated times (0.5, 2 and 5 min). Percentages of Citrine-positive cells are shown in the graph in Fig. 2a. b, Same as a, but with CRY omitted to test for proximity-dependence of TEVΔ–TEVcs interaction (cells express TEVΔ-mCherry instead of CRY-TEVΔ-mCherry). Each plot represents two replicates; n = 10,000 cells.

Supplementary Figure 4 TEV purification and kinetics.

Related to Figs. 2c,d and 3e,f. a, SDS–PAGE (9%) of purified TEV proteases. TEVΔ is 25 kD. MBP–TEV (full-length) is 75 kD. b, Michaelis–Menten plots for wild-type full-length TEV and uTEV3 (containing mutations I138T, S135N and T180A). Reactions were assembled with 100 nM purified protease and variable amounts (0.0075–0.32 mM) of substrate protein MBP–TEVcs(ENLYFGS)–GFP at 30 °C. Initial proteolysis rates were determined for each starting substrate concentration, using the in-gel fluorescence assay shown in Figs. 2c and 3e. Data were fit to a Michaelis–Menten enzyme-kinetics model with center values representing the mean and error bars representing the s.d. of three technical replicates.

Supplementary Figure 5 Profiling the sequence specificity of TEVΔ variants in yeast.

Related to Fig. 2f,g. a, Assay for profiling yeast sequence specificity. The protease variant of interest is co-expressed with a library of TEVcs sequences (flanked by LexA-VP16 TF at the C-terminal end, and a plasma-membrane anchor, mCherry, CIBN and LOV at the N-terminal end). Upon exposure of cells to blue 450 nm light, the CRY–CIBN interaction brings the protease proximal to TEVcs, and the LOV domain changes conformation to expose TEVcs. Sequences sensitive to TEV proteolysis will release the TF, which translocates to the nucleus and drives expression of the reporter gene Citrine. b, Sequence profile of the seven TEVcs libraries with randomized nucleotides (pooled together) before sorting. Each of the seven TEVcs libraries is randomized at a single position only. c, Analysis of single randomized positions in the TEV cleavage site, using wild-type TEVΔ, uTEV1Δ and uTEV2Δ. Sample FACS plots 6 h after blue-light exposure. Each plot represents one replicate; n = 10,000 cells. d, Viability assays in HEK 293T cells expressing the evolved TEV proteases. This experiment was performed once, with three biological replicates per sample. White dots indicate individual technical replicates.

Supplementary Figure 6 Optimizing the yeast platform for evolution of full-length high-affinity proteases.

Related to Fig. 3. a, To tune the dynamic range of the platform, the LexA DNA-binding domain was fused to one of three different transcriptional activators (TAs): VP16, B42 or Gal4. The constructs were expressed in yeast containing different numbers of LexA boxes upstream of the Citrine reporter gene. b, FACS data showing the effect of varying the number of LexA boxes in the promoter with different TAs. FACS data were collected 12 h following galactose induction. Each plot represents two replicates; n = 20,000 cells. c, Comparison of different full-length TEVs in the setup shown in Fig. 3a. The TEVcs was the high-affinity sequence ENLYFQ/S. FACS plots obtained 6 h after blue-light exposure for the indicated times (5, 10, 20 and 40 min). Each plot represents one replicate; n = 20,000 cells. Percentage of Citrine-positive cells in each condition used to generate the graph in Fig. 3b.

Supplementary Figure 7 Analysis of selected yeast populations and full-length TEV clones.

Related to Fig. 3. a, Same as Fig. 3c, but showing more conditions. b, Sequencing after round 3. 24 clones were sequenced and mutations found in each, relative to the original template uTEV1, are shown.

Supplementary Figure 8 Characterization of evolved full-length TEV mutants.

Related to Fig. 3d. a, Proteases were expressed in a yeast strain with 2 LexA boxes and high-affinity TEVcs (ENLYFQ/S) (configuration shown in Fig. 3a). FACS analysis performed 6 h after light irradiation. Each condition repeated once. n = 20,000 cells. b, Same as a with additional TEV mutants and additional conditions. The first 3 columns show shorter protein induction times in the dark (standard induction time is 12 h). Right 3 columns show cells 6 h following blue-light irradiation for the indicated times. Each condition performed twice. n = 20,000 cells.

Supplementary Figure 9 Profiling the sequence specificity of full-length TEV mutants in yeast.

Same assay as in Fig. 2f. FACS analysis performed 12 h after galactose induction. Each condition performed once; n = 20,000 cells.

Supplementary Figure 10 Comparison of evolved TEVs with TEV mutants.

TEVs were from Iverson et al.17, Bottomley et al.30 and Waugh et al.41. a, Side-by-side comparison in yeast, with full-length proteases and the high-affinity TEVcs (ENLYFQ/S). First four columns show yeast induced with galactose in the dark for 6.5 to 18 h before FACS analysis. Last two columns were irradiated with light before FACS analysis 6 h later. b, Side-by-side comparison of truncated proteases using the low-affinity TEVcs (ENLYFQ/M). FACS analysis was performed 6 h after blue-light exposure for the indicated times. Each condition was repeated once; n = 20,000 cells.

Supplementary Figure 11 Testing evolved TEV mutants in FLARE.

Related to Fig. 4e. a, HEK 293T cells were transiently transfected with FLARE constructs (as in Fig. 4a) incorporating the indicated TEV protease. Stimulation was performed using 5 mM CaCl2 and ionomycin for 30 s in the presence of blue light (467 nm, 60 mW per cm2, 10% duty cycle (0.5 s light every 5 s). Nine hours later, cells were fixed and imaged. This experiment was performed independently two times with similar results. b, Same experiment as in a but with luciferase as the reporter instead of mCherry. Stimulation times varied from 30 s to 5 min. This experiment was performed once with three technical replicates per condition. c, Comparison of uTEV1Δ with the truncated version of Iverson’s TEV in the context of FLARE. Cells were stimulated and analyzed as in b. This experiment was performed once, with three technical replicates per condition. d, Samples from c were imaged by confocal microscopy to confirm protease expression. The GFP channel is shown. Scale bar, 10 μm.

Supplementary Figure 12 Evaluation of uTEV1Δ in the context of FLARE in neurons.

Related to Fig. 4f. a, Rat cortical neurons were transduced at day 12 with FLARE constructs (packaged into AAV1/2 viruses), containing either the original TEVΔ protease or our evolved uTEV1Δ protease. At day 18 in vitro (DIV18), we stimulated the neurons using either field stimulation (3-s trains consisting of 32 1-ms 50-mA pulses at 20 Hz for a total of 1 or 5 min), or via replacement of culture medium with medium of identical composition (this mechanically stimulates the cultures and also provides a fresh source of glutamate). Light source was 467 nm, 60 mW per cm2, 10% duty cycle (0.5 s light every 5 s). Imaging was performed 18 h later. This experiment was replicated three times for each condition. Scale bars, 10 μm. b, Quantitation of data from a. Signal ratios were calculated from mean mCherry and mean GFP intensities across >50 cells per FOV. 10 FOVs were quantified per condition. Red lines indicates the mean of 10 FOVs.

Supplementary Figure 13 Testing the evolved protease uTEV1Δ in SPARK.

Related to Fig. 4g. HEK293T cells were transiently transfected with SPARK constructs (Fig. 4b) containing the indicated protease variant. Cells were stimulated with 10 μM isoproterenol for 60 s in the presence or absence of blue light (467 nm, 60 mW per cm2, 10% duty cycle (0.5s light every 5 s)). Nine hours later, cells were imaged. This experiment was replicated two times. Scale bars, 10 μm.

Supplementary Figure 14 Testing a different protease (TVMV) in the yeast platform.

Constructs were designed as in Fig. 1a, but TEV was replaced with TVMV protease, and TEVcs was replaced with the TVMV substrate sequences shown at right. FACS plots were collected 6 h after blue-light irradiation for the indicated times. Percentages give the fraction of Citrine-positive cells. Each plot is representative of two replicates; n = 20,000 cells.

Supplementary Figure 15 Selections to alter the sequence-specificity of TEV.

a,b, Analysis of full-length TEV libraries after 3 rounds of sorting against mutated TEV cleavage sequences: H at the P3 position of TEVcs in a, and W in the P3 position of TEVcs in b. The configuration of constructs was the same as in Fig. 3a. FACS plots were obtained 6 h after blue-light exposure for the indicated times. Percentages quantify Citrine-positive cells. This experiment was performed once; n = 10,000 cells. c,d, Sequencing after round 3. 24 clones were sequenced from each selection and mutations found in each, relative to the original template wild-type TEV, are shown.

Supplementary Figure 16 Characterization of specificity-altered TEV mutants in yeast.

Related to Supplementary Fig. 15. a,b, The indicated TEV mutants were compared to wild-type full-length TEV, using the altered TEVcs substrates P3 = H (a) and P3 = W (b). FACS plots obtained 6 hours after blue-light exposure for the indicated times. Percentages reflect the fraction of Citrine-positive cells. Each plot representative of two replicates; n = 10,000 cells.

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Sanchez, M.I., Ting, A.Y. Directed evolution improves the catalytic efficiency of TEV protease. Nat Methods 17, 167–174 (2020).

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