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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Additional data beyond that provided in the Figures and Supplementary Information are available from the corresponding author upon request.
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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).
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).
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.
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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.
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.
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.
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.
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.
Same assay as in Fig. 2f. FACS analysis performed 12 h after galactose induction. Each condition performed once; n = 20,000 cells.
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.
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.
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.
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.
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.
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.
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). https://doi.org/10.1038/s41592-019-0665-7
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