Facile Site-Specific Multiconjugation Strategies in Recombinant Proteins Produced in Bacteria

Merten, Hannes; Schaefer, Jonas V.; Brandl, Fabian; Zangemeister‐Wittke, Uwe; Plückthun, Andreas

doi:10.1007/978-1-4939-9654-4_17

Cited by 7 publications

(8 citation statements)

References 57 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…DBCO peptides can be easily conjugated to azide-functionalized nanoparticles, and this method will enable larger and more sequence-diverse DBCO peptides to be incorporated. Additionally, several methods exist to incorporate azides into proteins via engineered tRNA synthetases or Met replacement with azide-containing analogs, − enabling DBCO peptides to tag recombinant azido-proteins for various applications (e.g., proteome labeling and imaging). Although cyclooctynes can be genetically encoded into proteins, ,− these methods are more challenging to implement compared to azide .…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, several methods exist to incorporate azides into proteins via engineered tRNA synthetases or Met replacement with azide-containing analogs, − enabling DBCO peptides to tag recombinant azido-proteins for various applications (e.g., proteome labeling and imaging). Although cyclooctynes can be genetically encoded into proteins, ,− these methods are more challenging to implement compared to azide . The finding that (MeCN) 4 CuBF 4 prevents acid-catalyzed 5- endo - dig cycloisomerization in DBCO, and perhaps other alkyne-derived isomerization reactions, is an important observation.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

et al. 2021

View full text Add to dashboard Cite

The scope of proteins accessible to total chemical synthesis via native chemical ligation (NCL) is often limited by slow ligation kinetics. Here we describe Click-Assisted NCL (CAN), in which peptides are incorporated with traceless "helping hand" lysine linkers that enable addition of dibenzocyclooctyne (DBCO) and azide handles. The resulting strain-promoted alkyne−azide cycloaddition (SPAAC) increases their effective concentration to greatly accelerate ligations. We demonstrate that copper(I) protects DBCO from acid-mediated rearrangement during acidic peptide cleavage, enabling direct production of DBCO synthetic peptides. Excitingly, triazole-linked model peptides ligated rapidly and accumulated little side product due to the fast reaction time. Using the E. coli ribosomal subunit L32 as a model protein, we further demonstrate that SPAAC, ligation, desulfurization, and linker cleavage steps can be performed in one pot. CAN is a useful method for overcoming challenging ligations involving sterically hindered junctions. Additionally, CAN is anticipated to be an important stepping stone toward a multisegment, one-pot, templated ligation system.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

et al. 2021

View full text Add to dashboard Cite

show abstract

“…95 DBCO peptides can be easily conjugated to azide-functionalized nanoparticles, 82 and this method will enable larger and more sequence-diverse DBCO peptides to be incorporated. Additionally, several methods exist to incorporate azides into proteins via engineered tRNA synthetases or Met replacement with azide-containing analogs, [96][97][98][99][100] enabling DBCO peptides to tag recombinant azido-proteins for various applications (e.g., proteome labeling and imaging). Although cyclooctynes can be genetically encoded into proteins, 98,[100][101][102][103][104] these methods are more challenging to implement compared to azide.…”

Section: Click-assisted Ncl Of the E Coli 50s Ribosomal Subunit L32mentioning

confidence: 99%

“…Additionally, several methods exist to incorporate azides into proteins via engineered tRNA synthetases or Met replacement with azide-containing analogs, [96][97][98][99][100] enabling DBCO peptides to tag recombinant azido-proteins for various applications (e.g., proteome labeling and imaging). Although cyclooctynes can be genetically encoded into proteins, 98,[100][101][102][103][104] these methods are more challenging to implement compared to azide. 98 The finding that (MeCN)4CuBF4 prevents acid-catalyzed 5-endo-dig cycloisomerization in DBCO, and perhaps other alkyne-derived isomerization reactions, 105 is an important observation.…”

Section: Click-assisted Ncl Of the E Coli 50s Ribosomal Subunit L32mentioning

confidence: 99%

“…Although cyclooctynes can be genetically encoded into proteins, 98,[100][101][102][103][104] these methods are more challenging to implement compared to azide. 98 The finding that (MeCN)4CuBF4 prevents acid-catalyzed 5-endo-dig cycloisomerization in DBCO, and perhaps other alkyne-derived isomerization reactions, 105 is an important observation.…”

Section: Click-assisted Ncl Of the E Coli 50s Ribosomal Subunit L32mentioning

confidence: 99%

See 1 more Smart Citation

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

Erickson¹,

Fulcher²,

Kay³

2020

Preprint

View full text Add to dashboard Cite

<div><div><div><p>Chemoselective ligation reactions, such as native chemical ligation (NCL), enable the assembly of synthetic peptides into proteins. However, the scope of proteins accessible to total chemical synthesis is limited by ligation efficiency. Sterically hindered thioesters and poorly soluble peptides can undergo incomplete ligations, leading to challenging purifications with low yields. This work describes a new method, ClickAssisted NCL (CAN), which overcomes these barriers. In CAN, peptides are modified with traceless “helping hand” lysine linkers that enable addition of dibenzocyclooctyne (DBCO) and azide handles for strain-promoted alkyne-azide cycloaddition (SPAAC) reactions. This cycloaddition templates the peptides to increase their effective concentration and greatly accelerate ligation kinetics. After ligation, mild hydroxylamine treatment tracelessly removes the linkers to afford the native ligated peptide. Although DBCO is incompatible with standard Fmoc solid-phase peptide synthesis (SPPS) due to an acid-mediated rearrangement that occurs during peptide cleavage, we demonstrate that copper(I) protects DBCO from this side reaction, enabling direct production of DBCO-containing synthetic peptides. Excitingly, low concentrations of triazole-linked model peptides reacted ~1,200-fold faster than predicted for non-templated control ligations, which also accumulated many side products due to the long reaction time. Using the E. coli ribosomal subunit L32 as a model protein, we further demonstrate that the SPAAC, ligation, desulfurization, and linker cleavage steps can be performed in a one-pot fashion. CAN will be useful for overcoming ligation challenges to expand the reach of chemical protein synthesis.</p></div></div></div>

show abstract

Bioconjugation

2019

Methods in Molecular Biology

View full text Add to dashboard Cite

concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

show abstract

Facile Site-Specific Multiconjugation Strategies in Recombinant Proteins Produced in Bacteria

Cited by 7 publications

References 57 publications

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I)

Bioconjugation

Contact Info

Product

Resources

About