Generation of a Genetically Encoded, Photoactivatable Intein for the Controlled Production of Cyclic Peptides

Abstract

Cyclic peptides are important natural products and hold great promise for the identification of new bioactive molecules. The split-intein-mediated SICLOPPS technology provides a generic access to fully genetically encoded head-to-tail cyclized peptides and large libraries thereof (SICLOPPS=split-intein circular ligation of peptides and proteins). However, owing to the spontaneous protein splicing reaction, product formation occurs inside cells, making peptide isolation inconvenient and precluding traditional in vitro assays for inhibitor discovery. The design of a genetically encoded, light-dependent intein using the photocaged tyrosine derivative ortho-nitrobenzyltyrosine incorporated at an internal, non-catalytic position is now reported. Stable intein precursors were purified from the E.coli expression host and subsequently subjected to light activation in vitro for both the regular protein splicing format and cyclic peptide production, including the natural product segetalin H as an example. The activity of the intein could also be triggered in living cells.

Building Peptide Bonds in Haifa: The Seventh Chemical Protein Synthesis (CPS) Meeting

“…Henning Mootz (University of Münster, Germany) presented a different approach for linking peptides and protein sequences: the use of split inteins as a chemoenzymatic tool to build peptide bonds through trans ‐splicing. His lab has reported a set of useful split inteins over the recent years, including the natural Gp41‐1 intein, the fastest known trans ‐splicing intein, and an engineered, photoactivatable intein for light‐dependent protein splicing . Mootz also discussed mechanistic insights into the protein splicing pathway that could facilitate future intein applications.…”

“…His lab has reported as et of usefuls plit inteinso ver the recent years, including the natural Gp41-1 intein, the fastest known trans-splicing intein, [19] and an engineered, photoactivatable intein for light-dependent protein splicing. [20] Mootz also discussed mechanistic insights into the protein splicing pathway that could facilitatef uture intein applications. Yeta nother biochemical pathway to generate peptides was discussed by Donald Hilvert (ETH Zürich, Switzerland).…”

Modular protein domains: an engineering approach toward functional biomaterials

“…For example, both cysteine and serine residues in inteins have been modified with a photocage, and these photoactivatable inteins are promising protein-labeling tools with exquisite spatiotemporal control that can be directly used in live mammalian cells [52,53]. Inteins with photoactive tyrosines have also been used as a tool for making cyclic peptides [54]. …”

67th Mosbacher Kolloquium: Protein Design: From First Principles to Biomedical Applications

“…Using directed evolution, he obtained novel engineered inteins with temperature‐independent activity and rapid catalysis. By means of chemical modification, a photocaged, engineered intein was developed, activated by light to induce protein splicing …”