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.

我们报告了通过利用细胞内蛋白质反式剪接结合高效的分裂内含蛋白，在大肠杆菌细胞内高活性的生物活性θ-防御素RTD-1的异源表达。RTD-1是具有三个二硫键和炭疽致死因子蛋白酶的天然抑制剂的小型骨架环化多肽。重组RTD-1被天然折叠并能够抑制炭疽致死因子蛋白酶。RTD-1在细胞内的表达非常有效，每克湿大肠杆菌细胞产生约0.7 mg折叠的RTD-1 。该方法用于在活大肠杆菌中生成基于基因编码的基于RTD-1的肽库细胞。这些结果清楚地证明了在活的大肠杆菌细胞中使用基于基因编码的基于RTD-1的肽库的可能性，这是开发使用环肽RTD-1作为细胞内筛选和定向进化技术的关键的第一步。分子支架。