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PROTein Evolution Using Selection (PROTEUS) [Nanopore]

ABSTRACT: Directed evolution (DE) is a process of mutation and iterative artificial selection to breed biomolecules with new or improved activity. DE platforms are primarily prokaryotic or yeast-based, and stable highly mutagenic mammalian systems have been challenging to establish and apply. To this end, we developed PROTein Evolution Using Selection (PROTEUS), a new platform that uses chimeric virus-like vesicles (VLVs) to enable extended mammalian DE campaigns without loss of system integrity. This platform, consisting of a minimal modified Semliki Forest virus genome controlling expression of the Indiana vesiculovirus G coat protein, is stable and can generate sufficient diversity for DE in mammalian systems. Using PROTEUS, we altered the doxycycline responsiveness of tetracycline-controlled transactivators, generating a more sensitive TetON-4G tool for gene regulation. PROTEUS is also compatible with intracellular nanobody evolution, and we use it to design a novel DNA damage-responsive anti-p53 nanobody. Overall, PROTEUS is a robust, efficient, and stable platform to direct evolution of biomolecules within mammalian cells.

ORGANISM(S): Semliki Forest virus

PROVIDER: GSE250501 | GEO | 2025/04/03

REPOSITORIES: GEO

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