In-cell production of a genetically-encoded library based on the θ-defensin RTD-1 using a bacterial expression system. Issue 6 (15th March 2018)

• Record Type: Journal Article
• Authors: Bi, Tao; Li, Yilong; Shekhtman, Alexander; Camarero, Julio A.
• Is Part Of: Bioorganic & medicinal chemistry. Volume 26:Issue 6(2018)
• Page Start: 1212
• Page End: 1219
• Publication Date: 2018-03-15
• Subjects: RTD-1 -- θ-Defensins -- Anthrax lethal factor -- Genetically-encoded libraries -- Backbone-cyclized peptides -- Protein splicing -- Split-intein; Bioorganic chemistry -- Periodicals; Pharmaceutical chemistry -- Periodicals; Biochemistry -- Periodicals; Chemistry, Clinical -- Periodicals; Chemistry, Organic -- Periodicals; Chimie bio-organique -- Périodiques; Chimie pharmaceutique -- Périodiques; 615.19
• DOI: 10.1016/j.bmc.2017.09.002
• Languages: English
• ISSNs: 0968-0896

Abstract

Graphical abstract: Abstract: 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.