Synthesis of Non-linear Protein Dimers through a Genetically Encoded Thiol-ene Reaction

Torres‐Kolbus, Jessica; Chou, Chin Cheng; Liu, Jihe; Deiters, Alexander

doi:10.1371/journal.pone.0105467

Cited by 15 publications

(10 citation statements)

References 100 publications

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“…Besides protection, allyl sulfides are expected to enable controlled bioorthogonal protein modification such as photo‐induced thiol‐ene coupling . For these reasons, Sac has been described as a “privileged” ncAA for post‐translational protein‐labeling chemistry, as it offers unprecedented chemical versatility combined with small size, thus reducing the risk of interference with the modified protein's structure and function.…”

Section: Figurementioning

confidence: 99%

Design of S‐Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl‐tRNA Synthetase Variant

Exner

Kuenzl

et al. 2016

ChemBioChem

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The noncanonical amino acid S-allyl cysteine (Sac) is one of the major compounds of garlic extract and exhibits ar ange of biological activities. It is also as mall bioorthogonal alkene tag capable of undergoing controlled chemical modifications, such as photoinduced thiol-ene coupling or Pd-mediated deprotection. Its small size guarantees minimal interference with protein structure and function. Here, we report as imple protocol efficiently to couple in-situ semisynthetic biosynthesis of Sac and its incorporation into proteins in response to amber (UAG) stop codons. We exploitedt he exceptional malleability of pyrrolysyl-tRNA synthetase (PylRS) and evolved an S-allylcysteinyltRNA synthetase (SacRS) capable of specifically acceptingt he small, polar amino acid instead of its long andb ulky aliphatic natural substrate. We succeeded in generating an ovel and inexpensive strategy for the incorporation of af unctionally versatile amino acid. This will help in the conversiono fo rthogonal translation from as tandard technique in academic research to industrial biotechnology.

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Section: Figurementioning

confidence: 99%

Design of S‐Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl‐tRNA Synthetase Variant

Exner

Kuenzl

et al. 2016

ChemBioChem

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“…This specific coupling reaction is initiated by irradiation at 365 nm in aqueous conditions in the presence of photo-initiator for max. 15 min [40].…”

Section: Resultsmentioning

confidence: 99%

In-Cell Synthesis of Bioorthogonal Alkene Tag S-Allyl-Homocysteine and Its Coupling with Reprogrammed Translation

Nojoumi

Schwagerus

et al. 2019

IJMS

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In this study, we report our initial results on in situ biosynthesis of S-allyl-l-homocysteine (Sahc) by simple metabolic conversion of allyl mercaptan in Escherichia coli, which served as the host organism endowed with a direct sulfhydration pathway. The intracellular synthesis we describe in this study is coupled with the direct incorporation of Sahc into proteins in response to methionine codons. Together with O-acetyl-homoserine, allyl mercaptan was added to the growth medium, followed by uptake and intracellular reaction to give Sahc. Our protocol efficiently combined the in vivo synthesis of Sahc via metabolic engineering with reprogrammed translation, without the need for a major change in the protein biosynthesis machinery. Although the system needs further optimisation to achieve greater intracellular Sahc production for complete protein labelling, we demonstrated its functional versatility for photo-induced thiol-ene coupling and the recently developed phosphonamidate conjugation reaction. Importantly, deprotection of Sahc leads to homocysteine-containing proteins—a potentially useful approach for the selective labelling of thiols with high relevance in various medical settings.

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“…A20FMDV2 was conjugated to ssDNA via a maleimidethiol reaction, bridging the cysteine thiol on the peptide and a deprotected maleimide on ssDNA (5' end OH group of the phosphate). The cRFDfC peptides were conjugated to ssDNA with a 1 hour, UV mediated thiol-ene reaction 35 , via a sulfhydryl group on the peptide and a thiol group on ssDNA modified with an acrydite moiety. For all ssDNA-peptide products, Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) was used to verify successful conjugation.…”

Section: Synthesis and Assembly Of Functionalised Dna Origami Nanostrmentioning

confidence: 99%

Probing the nanoscale organisation and multivalency of cell surface receptors: DNA origami nanoarrays for cellular studies with single-molecule control

et al. 2019

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Synthesis of Non-linear Protein Dimers through a Genetically Encoded Thiol-ene Reaction

Cited by 15 publications

References 100 publications

Design of S‐Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl‐tRNA Synthetase Variant

Design of S‐Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl‐tRNA Synthetase Variant

In-Cell Synthesis of Bioorthogonal Alkene Tag S-Allyl-Homocysteine and Its Coupling with Reprogrammed Translation

Probing the nanoscale organisation and multivalency of cell surface receptors: DNA origami nanoarrays for cellular studies with single-molecule control

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