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

Inventors

Ting, Alice Y. • SANCHEZ LOPEZ, MATEO ISIDRO

Assignees

Leland Stanford Junior University • CZ Biohub SF LLC • Biohub

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.

Core Innovation

The invention provides 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. The library includes yeast cells containing a first fusion protein and a second fusion protein, along with a transcription factor and a reporter gene whose transcription depends on the transcription factor. Different yeast cells contain different TEV protease variants as part of the first fusion protein, enabling protease activity of each variant to be read out through reporter expression.

In the yeast-cell system, the first fusion protein includes a first member of a photoinducible protein binding pair and a TEV protease variant that cleaves a proteolytically cleavable linker. The second fusion protein includes a transmembrane domain, a second member of a photoinducible protein binding pair, and a light-oxygen-voltage-sensing (LOV) domain sequence, and it presents the proteolytically cleavable linker comprising a TEV cleavage sequence (TEVcs) together with a transcription factor. When the yeast cells are irradiated in culture with light that induces binding between the photoinducible components, the TEV protease variant cleaves the TEVcs-containing linker to drive expression of the transcription factor-dependent reporter gene in at least some yeast cells.

The selection is based on the use of TEV protease variants that each contain one or more amino acid changes compared with a C-terminally truncated wild-type TEV protease having SEQ ID NO: 2. By irradiating the yeast cells so that light-induced binding couples protease activity to reporter gene expression, the library enables selection of TEV protease variants exhibiting altered catalytic properties resulting from directed evolution of the TEV protease template.

Claims Coverage

The independent claim defines a yeast-cell library for light-induced, reporter-coupled selection of modified TEV protease variants whose catalytic properties are altered by directed evolution. It includes two fusion proteins built from photoinducible binding components, a TEV cleavage linker, a transcription factor, and a reporter gene, with different yeast cells containing different TEV protease variants versus a C-terminally truncated wild-type template. The claim includes two inventive features.

Across the independent claim, light-induced binding of photoinducible fusion protein components triggers TEVcs linker cleavage by TEV protease variants. The resulting protease activity is coupled to transcription factor-dependent reporter gene expression, enabling selection among yeast cells carrying different TEV protease variants relative to the C-terminally truncated wild-type TEV protease (SEQ ID NO: 2).

Stated Advantages

Documented Applications

No documented applications found