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

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.