Bioorganic and Medicinal Chemistry
Bioorganic and Medicinal Chemistry
5 years ago

In-cell production of a genetically-encoded library based on the θ-defensin RTD-1 using a bacterial expression system

In-cell production of a genetically-encoded library based on the θ-defensin RTD-1 using a bacterial expression system
We report the high-yield heterologous expression of bioactive θ -defensin RTD-1 inside Escherichia coli cells by making use of intracellular protein trans-splicing in combination with a high efficient split-intein. RTD-1 is a small backbone-cyclized polypeptide with three disulfide bridges and a natural inhibitor of anthrax lethal factor protease. Recombinant RTD-1 was natively folded and able to inhibit anthrax lethal factor protease. In-cell expression of RTD-1 was very efficient and yielded ≈ 0.7 mg of folded RTD-1 per gram of wet E. coli cells. This approach was used to generate of a genetically-encoded RTD-1-based peptide library in live E. coli cells. These results clearly demonstrate the possibility of using genetically-encoded RTD-1-based peptide libraries in live E. coli cells, which is a critical first step for developing in-cell screening and directed evolution technologies using the cyclic peptide RTD-1 as a molecular scaffold.

Publisher URL: www.sciencedirect.com/science

DOI: S096808961731516X

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