We present a platform for the continuous directed evolution of proteins in human cells. This platform is widely adaptable, such that we can theoretically evolve any protein whose function is linked to adenoviral propagation. By using replication-deficient strains of adenovirus, we have created a safe platform for protein evolution in human cells. Since adenovirus can infect many different cell types, this platform can be implemented in a variety of different human cellular environments. In a proof-of-concept experiment, we evolved a doxycycline-insensitive variant of the tetracycline transactivator. Additionally, we envision the evolution of other important biomolecules such as transcription factors, proteases, endonucleases and monobodies. We plan on studying the evolution of protein mutants that lead to cancer proliferation and chemotherapeutic resistance. Additionally, we will create tools to perturb and interrogate signaling pathways unique to metazoan cells. We will also explore the role of the proteostasis network on evolutionary trajectories, studying whether increased protein folding capacity allows for the proper folding of more active variants. This adaptable, safe and cell-type independent platform for directed evolution will provide researchers the ability to generate useful biochemical tools and therapeutics that will reliably function in human cells.

Evolving biomolecules through a human cell based continuous directed evolution platform

256th National Meeting (2018)

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