Biosynthesis and Biological Screening of a Genetically Encoded Library Based on the Cyclotide MCoTI‐I

Abstract

Cell‐ing point: This study shows that MCoTI‐cyclotides can provide an ideal scaffold for the biosynthesis of large combinatorial libraries inside living E. coli cells. Coupled to an appropriate in vivo reporter system, this library may rapidly be screened, for example, by fluorescence‐activated cell sorting.

Phosphatidylethanolamine Binding Is a Conserved Feature of Cyclotide-Membrane Interactions

“…In particular, their native pharmacological activities might be used in the development of uterotonic or antimicrobial agents, or the cyclotide scaffold could in principle be used as an inert framework to stabilize linear bioactive peptide epitopes (11). Indeed, the use of cyclotides as a scaffold for drug design has been explored both using synthetic chemistry (12,13) and through biosynthesis of a genetically encoded cyclotide combinatorial library expressed inside Escherichia coli (14). The bioengineering of such libraries enables the rapid screening and the selection of new sequences with specific biological activities and high stability (15).…”

Biological Activities of Natural and Engineered Cyclotides, a Novel Molecular Scaffold for Peptide-Based Therapeutics

“…Trypsin inhibitor cyclotides share a high sequence homology with related cystine-knot trypsin inhibitors found in squash such as EETI-II (Ecballium elaterium trypsin inhibitor II), and in fact can be considered cyclized homologs of these protease inhibitors. Thus, cyclotides can be considered natural combinatorial peptide libraries structurally constrained by the cystineknot scaffold [23] and head-to-tail cyclization but are permissive of hypermutation of essentially all residues with the exception of the strictly conserved cysteines that comprise the knot [24][25][26][27].…”

“…3) (see reference [68] for a recent review). This method can generate folded cyclotides either in vivo or in vitro using standard bacterial expression systems [26,69,70]. Inteins are internal selfprocessing domains that undergo post-translational processing to splice together flanking external domains (exteins) [71].…”

“…More recently, we have also reported the biosynthesis of a genetically encoded library of MCoTI-I based cyclotides in E. coli cells [26]. The cyclization/folding of the library was performed either in vitro, by incubation with a redox buffer containing glutathione, or by in vivo self-processing of the corresponding precursor proteins.…”

“…The cyclization/folding of the library was performed either in vitro, by incubation with a redox buffer containing glutathione, or by in vivo self-processing of the corresponding precursor proteins. The bacterial gyrase A intein from Mycobacterium xenopus was used in this work [26]. This intein typically express at higher yields than the yeast VMA intein in E. coli expression systems [26].…”