Efficient recombinant expression of SFTI-1 in bacterial cells using intein-mediated protein trans-splicing.

We report for the first time the recombinant expression of bioactive wild-type sunflower trypsin inhibitor 1 (SFTI-1) inside E. coli cells by making use of intracellular protein trans-splicing in combination with a high efficient split-intein. SFTI-1 is a small backbone-cyclized polypeptide with a single disulfide bridge and potent trypsin inhibitory activity. Recombinantly produced SFTI-1 was fully characterized by NMR and was observed to actively inhibit trypsin. The in-cell expression of SFTI-1 was very efficient reaching intracellular concentration ≈ 40 µM. This study clearly demonstrates the possibility of generating genetically encoded SFTI-based peptide libraries in live E. coli cells, and is a critical first step for developing in-cell screening and directed evolution technologies using the cyclic peptide SFTI-1 as a molecular scaffold.