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-totail 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, lightdependent intein using the photocaged tyrosine derivative ortho-nitrobenzyltyrosine incorporated at an internal, noncatalytic 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.

Specific Ligation of Two Multimeric Enzymes with Native Peptides and Immobilization with Controlled Molar Ratio

“…DAAO&CAT exhibits a K m value much smaller than that of DAAO, indicating that the spliced product DAAO&CAT has significantly improved the affinity toward the substrate. 40 The K cat /K m ratio has been used to measure catalysis efficiency. 40 The K cat /K m ratio of DAAO&CAT is 22.4 times that of DAAO, indicating a much higher catalytic efficiency achieved by DAAO&CAT.…”

“…40 The K cat /K m ratio has been used to measure catalysis efficiency. 40 The K cat /K m ratio of DAAO&CAT is 22.4 times that of DAAO, indicating a much higher catalytic efficiency achieved by DAAO&CAT. This kinetics result is in accordance with the qualitative comparison as shown in Figure 5a.…”

Advancing the Frontiers of Chemical Protein Synthesis—The 7th CPS Meeting, Haifa, Israel

“…Christian Hackenberger (FMP and Humboldt University Berlin, Germany) described the application of chemoselective reactions for site-specific labeling and cellular uptake of antibodies (Herce et al, 2017; Schumacher et al, 2017a; Schumacher et al, 2017b). Recent advances in intein-mediated ligation were presented by Henning Mootz (University of Münster, Germany), who provided mechanistic insights into the protein splicing pathway and introduced a set of useful split inteins, including the natural Gp41-1 intein as the fastest known trans-splicing intein (Carvajal-Vallejos et al, 2012) and an engineered, photoactivatable intein for light-dependent protein splicing (Boecker et al, 2015). Marcel Schmidt (EnzyPep, University of Amsterdam, Netherlands) presented recent developments on peptiligases with broad and narrow specificities for protein ligation and labeling (Nuijens et al, 2016; Schmidt et al, 2017; Toplak et al, 2016).…”

Recent advances in the optical control of protein function through genetic code expansion

“…One limitation of the approach is the necessity of a cysteine residue at the splice site; which, if not naturally present, will leave a scar following intein excision. In addition, optically controlled inteins have been applied to protein splicing in yeast [33] and generation of cyclic peptides in E. coli [34].…”