IP Library › Granted Patent US 12,590,325
Granted Patent B2
US 12,590,325 · App. 17/642,635 · Granted Mar 31, 2026

Directed evolution for obtaining improved variants of TEV protease for biotechnological applications

Inventors: Alice Y. Ting (Palo Alto, CA); Mateo Isidro Sanchez Lopez (Menlo Park, CA)
Assignees: CZ Biohub SF, LLC; The Board of Trustees of the Leland Stanford Junior University
C12P21/02C12N9/506C12N15/1086C12N15/625C12Y304/22044
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Quick Facts
Patent No.
US 12,590,325
App. No.
17/642,635
Granted
Mar 31, 2026
Kind
B2
Abstract

Tobacco etch virus protease (TEV) is one of the most widely used proteases in biotechnology because of its exquisite sequence-specificity. A limitation of TEV is its slow catalytic rate, which limits product generation and therefore signal output. Provided is a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is determined via proteolytic release of a membrane-anchored transcription factor, and access to TEV's cleavage site is temporally regulated using a photosensory LOV domain. By gradually decreasing light exposure time, faster variants of TEV were selected over multiple rounds of selection. The mutant TEV proteases and the directed evolution platform are useful in a wide range of biotechnology applications, such as FLARE and SPARK tools.

Claims (41)

1 . A library of yeast cells for selecting a modified Tobacco Etch Virus (TEV) protease having altered catalytic properties consequent to directed evolution of a TEV protease template, wherein the library comprises yeast cells that each contain the following:

(a) a first fusion protein comprising:

(i) a first member of a photoinducible protein binding pair;

(ii) a TEV protease variant that cleaves a proteolytically cleavable linker;

(b) a second fusion protein comprising:

(i) a transmembrane domain;

(ii) a second member of a photoinducible protein binding pair;

(iii) a light-oxygen-voltage-sensing (LOV) domain sequence;

(iv) said proteolytically cleavable linker, comprising a TEV cleavage sequence (TEVcs);

(v) a transcription factor; and

(c) a reporter gene that is transcribed by the transcription factor;

wherein different yeast cells in the library contain different TEV protease variants as part of the first fusion protein;

wherein the different TEV protease variants each contain one or more amino acid changes compared with a C-terminally truncated wild-type TEV protease having SEQ ID NO: 2; and

wherein when the yeast cells are irradiated in culture with a light that induces binding of component (a) to component (b), then as a consequence, component (c) is expressed in at least some of the yeast cells as an indication of protease activity of the respective TEV protease variant contained therein.

2 . The yeast cell library of claim 1 , wherein the first member of the photoinducible protein binding pair is a cryptochrome (CRY) and the second member of the photoinducible protein binding pair is a cryptochrome-interacting basic-helix-loop-helix protein (CIB).

3 . The yeast cell library of claim 1 , wherein the LOV domain sequence comprises a C-terminal alpha helix fused to the TEV cleavage sequence.

4 . The yeast cell library of claim 1 , wherein the transmembrane domain comprises an amino acid sequence from pheromone alpha factor receptor (STE2) or a truncated STE2.

5 . The yeast cell library of claim 1 , wherein the first fusion protein, the second fusion protein, or both further comprise a fluorescent protein.

6 . The yeast cell library of claim 1 , wherein the TEV protease variant in each yeast cell in the library is a C-terminally truncated TEV protease.

7 . The yeast cell library of claim 1 , wherein the transcription factor comprises transactivating tegument protein VP16.

8 . The yeast cell library of claim 1 , wherein the TEV protease variant in the first fusion protein of each cell in the library has been generated by error-prone polymerase chain reaction (PCR).

9 . A method for identifying a modified TEV protease having altered catalytic properties, the method comprising:

(a) obtaining a library of yeast cells according to claim 1 ,

(b) irradiating yeast cells from the library in culture with light that induces binding of the first and the second member of the photoinducible protein binding pair to each other in individual yeast cells in the library;

(c) selecting yeast cells that express the reporter gene; and

(d) determining altered catalytic properties in the yeast cells selected in step (c) compared to a control protease, thereby identifying said modified TEV protease.

10 . The method of claim 9 , wherein the irradiating produces an intermolecular complex between the first and second members of the photoinducible protein binding pair and induces a conformational change in the LOV domain sequence to expose a protease substrate cleavage sequence to the TEV protease variant in each yeast cell.

11 . The method of claim 9 , wherein the first member of the photoinducible protein binding pair is a cryptochrome (CRY) and the second member of the photoinducible protein binding pair is a cryptochrome-interacting basic-helix-loop-helix protein (CIB).

12 . The method of claim 9 , wherein the selecting in step (c) comprises selecting yeast cells that express the reporter gene at levels at least one order of magnitude greater than yeast cells that do not express component (a).

13 . The method of claim 8 , wherein the irradiating step (b) and selecting of step (c) are repeated one or more times.

14 . The method of claim 13 , wherein the irradiating step (b) is done for a period of time, and the period of time is decreased when step (b) is repeated.

15 . The method of claim 14 , wherein a percentage of cells expressing the reporter gene increases each time the period of time in step (b) is decreased.

16 . The method of claim 9 , wherein step (a) comprises:

(i) generating a library of polynucleotide vectors that each encode a TEV protease variant by error-prone polymerase chain reaction (PCR) of a TEV protease template;

(ii) integrating a plurality of vectors from said library of polynucleotide vectors into plasmids encoding said first fusion protein; and

(iii) transforming a plurality of said plasmids into separate yeast cells to be expressed as part of an assembly of said first fusion protein, said second fusion protein, and said reporter gene;

thereby forming said yeast cell library.

17 . The method of claim 16 , wherein said TEV protease template is a low affinity TEV protease characterized as having a Km that is greater than 300 microMolar.

18 . The method of claim 9 , wherein the TEV protease variants identified in step (d) increased catalytic efficiency compared with C-terminally truncated wild-type TEV protease having SEQ ID NO:2, wherein catalytic efficiency is defined as the rate of proteolytic cleavage (k cat ).

19 . The method of claim 9 , wherein the TEV protease variants identified in step (d) have relaxed TEV sequence specificity compared with C-terminally truncated wild-type TEV protease having SEQ ID NO:2.

20 . The method of claim 9 , wherein one or more of the TEV protease variants identified in step (d) comprise a mutation or combination thereof selected from T30A, T301, S31W, S135F, 1138T, S153N, S153D, T180A, a double T30A/S153N mutation, atriple 1138T/S153N/T180A mutation, and a quadruple S135F/1138T/S153N/T180A mutation.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 25, 2022
From: THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY
To: CHAN ZUCKERBERG BIOHUB, INC.; THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY
Reel/Frame 059697/0808 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 14, 2022
From: TING, ALICE Y.; LOPEZ, MATEO ISIDRO SANCHEZ
To: THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY
Reel/Frame 059260/0236 →
Continuity (2)
Provisional Application 62906373 · Sep 26, 2019
Related Publication 20230067225A1 · Mar 2, 2023
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