In vivo biosynthesis of an Ala-scan library based on the cyclic peptide SFTI-1

Abstract

We present the in-vivo biosynthesis of wild-type sunflower trypsin inhibitor 1 (SFTI-1) inside E. coli cells using an intramolecular native chemical ligation in combination with a modified protein splicing unit. SFTI-1 is a small backbone cyclized polypeptide with a single disulfide bridge. A small library containing multiple Ala mutants was also biosynthesized and its activity was assayed using a trypsin-binding assay. This study clearly demonstrates the exciting possibility of generating large cyclic peptide libraries in live E. coli cells, and is a critical first step for developing in-vivo screening and directed evolution technologies using the cyclic peptide SFTI-1 as a molecular scaffold.

Prospects for the design of new therapeutically significant protease inhibitors based on knottins and sunflower seed trypsin inhibitor (SFTI 1)

“…Эти подходы подробно рассмотрены в ряде обзоров [43,85,93]. Первый подход был использован для получения гетерологической экспрессией в E. coli SFTI 1 и библиотеки его аналогов для аланинового скрининга [94]. В работе [95] [95].…”

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

“…These requirements usually depend on the type of split-intein used, and seriously limit the diversity of the libraries that can be generated when using small cyclic peptide templates. Our group has recently demonstrated the expression in E. coli of libraries based on the cyclic peptide SFTI-1 (a backbone cyclized Bowman-Birk trypsin inhibitor) using an intein-mediated backbone cyclization approach (see above), which allows the biosynthesis of backbone-cyclized polypeptides without any sequence requirement limitation [89].…”

“…This library was cyclized in vitro by incubation with a redox buffer containing reduced glutathione (GSH) as a thiol co-factor, thus mimicking the intracellular conditions, where GSH is the most abundant thiol co-factor. The use of GSH allows the cyclization and folding to happen in one step [69,89]. Analysis of the cyclization/folding reaction by HPLC and mass spectrometry revealed that all the members of the kalata B1 based library were expressed and processed with similar yields to give the corresponding natively folded cyclotides [69].…”

The science and business of novel therapeutics: the need for a complete picture

“…A wide range of chemical [54][55][56] and biological [57][58][59][60] studies on the synthesis of cyclotides have been carried out recently and these typically involve the formation of a head-to-tail cyclic backbone before disulfide bond formation, rather than the reverse, as is thought to occur in nature, that is, disulfide bond formation followed by cyclization via the AEP as described above. The closest mimic to the natural biosynthesis is perhaps a chemoenzymatic approach in which a linearized version of MCoTI-2 was chemically synthesized and then enzymatically cyclized using trypsin [61 ].…”

“…The relationship between the in vitro efficacy and the in vivo efficacy of antimicrobial agents remains unclear, and prediction of in vivo activity is very challenging, due to variations in potency that can occur due to many uncontrolled parameters in the host systems (58,59). Despite the challenges of using in vitro potency to predict in vivo potency, we found that the most potent peptide in the in vitro study, pYR, was also the most potent peptide in treating S. aureus skin infections in mice.…”