Chemischer “Bypass” des Intein‐katalysierten N‐S‐Acyltransfers im Proteinspleißen

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.

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

Böcker

Friedel

Matern

et al. 2014

Angewandte Chemie

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

et al. 2014

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View full text Add to dashboard Cite

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.

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Biochemical and Structural Characterization of an Unusual and Naturally Split Class 3 Intein

et al. 2020

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Split inteins are indispensable tools for protein engineering because their ligation and cleavage reactions enable unique modifications of the polypeptide backbone. Three different classes of inteins have been identified according to the nature of the covalent intermediates resulting from the acyl rearrangements in the multistep protein-splicing pathway. Class 3 inteins employ a characteristic internal cysteine for a branched thioester intermediate. A bioinformatic database search of nonredundant protein sequences revealed the absence of split variants in 1701 class 3 inteins. We have discovered the first reported split class 3 intein in a metagenomics data set and report its biochemical, mechanistic and structural analysis. The AceL NrdHF intein exhibits low sequence conservation with other inteins and marked deviations in residues at conserved key positions, including a variation of the typical class-3 WCT triplet motif. Nevertheless, functional analysis confirmed the class 3 mechanism of the intein and revealed excellent splicing yields within a few minutes over a wide range of conditions and with barely detectable cleavage side reactions. A high-resolution crystal structure of the AceL NrdHF precursor and a mutagenesis study explained the importance and roles of several residues at the key positions. Tolerated substitutions in the flanking extein residues and a high affinity between the split intein fragments further underline the intein's future potential as a ligation tool.

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Chemischer “Bypass” des Intein‐katalysierten N‐S‐Acyltransfers im Proteinspleißen

Cited by 3 publications

References 40 publications

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

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

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

Biochemical and Structural Characterization of an Unusual and Naturally Split Class 3 Intein

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