Chemical synthesis of Shiga toxin subunit B using a next-generation traceless “helping hand” solubilizing tag

Fulcher, James; Petersen, Matthew J.; giesler, riley; Cruz, Zachary S; Eckert, Debra M.; Francis, James N.; kawamoto, eric; Jacobsen, Michael T.; Kay, Michael S.

doi:10.1039/c9ob02012h

Cited by 15 publications

(11 citation statements)

References 50 publications

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“…STxB is obtained in amounts that are typical for proteins which are expressed in the periplasmic space. STxB has recently also been chemically synthesized and refolded in vitro [ 199 ]. Whether this type of material can also be used to obtain functional vaccine conjugates remains to be tested.…”

Section: Potential Limitations Of Stxbmentioning

confidence: 99%

STxB as an Antigen Delivery Tool for Mucosal Vaccination

Tartour

Johannes

2022

Toxins

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Immunotherapy against cancer and infectious disease holds the promise of high efficacy with minor side effects. Mucosal vaccines to protect against tumors or infections disease agents that affect the upper airways or the lung are still lacking, however. One mucosal vaccine candidate is the B-subunit of Shiga toxin, STxB. In this review, we compare STxB to other immunotherapy vectors. STxB is a non-toxic protein that binds to a glycosylated lipid, termed globotriaosylceramide (Gb3), which is preferentially expressed by dendritic cells. We review the use of STxB for the cross-presentation of tumor or viral antigens in a MHC class I-restricted manner to induce humoral immunity against these antigens in addition to polyfunctional and persistent CD4+ and CD8+ T lymphocytes capable of protecting against viral infection or tumor growth. Other literature will be summarized that documents a powerful induction of mucosal IgA and resident memory CD8+ T cells against mucosal tumors specifically when STxB-antigen conjugates are administered via the nasal route. It will also be pointed out how STxB-based vaccines have been shown in preclinical cancer models to synergize with other therapeutic modalities (immune checkpoint inhibitors, anti-angiogenic therapy, radiotherapy). Finally, we will discuss how molecular aspects such as low immunogenicity, cross-species conservation of Gb3 expression, and lack of toxicity contribute to the competitive positioning of STxB among the different DC targeting approaches. STxB thereby appears as an original and innovative tool for the development of mucosal vaccines in infectious diseases and cancer.

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Section: Potential Limitations Of Stxbmentioning

confidence: 99%

STxB as an Antigen Delivery Tool for Mucosal Vaccination

Tartour

Johannes

2022

Toxins

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“…Examples for internal strategies were published by Tsuda et al (2018a) using a side-chain polylysine tag to increase the solubility during desulfurization. Other examples include the addition of a polyarginine tag at the side chain of amyloid-beta (Aβ) peptides or polylysine tags over a Ddae-linker for the synthesis of the 97-residue co-chaperonin GroES (Fulcher et al, 2019). External conditions are usually based on the addition of cosolvents such as fluorinated alcohols to the desulfurization buffer.…”

Section: Structurementioning

confidence: 99%

Challenges and Perspectives in Chemical Synthesis of Highly Hydrophobic Peptides

Mueller

Baumruck

Zhdanova

et al. 2020

Front. Bioeng. Biotechnol.

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Solid phase peptide synthesis (SPPS) provides the possibility to chemically synthesize peptides and proteins. Applying the method on hydrophilic structures is usually without major drawbacks but faces extreme complications when it comes to "difficult sequences." These includes the vitally important, ubiquitously present and structurally demanding membrane proteins and their functional parts, such as ion channels, G-protein receptors, and other pore-forming structures. Standard synthetic and ligation protocols are not enough for a successful synthesis of these challenging sequences. In this review we highlight, summarize and evaluate the possibilities for synthetic production of "difficult sequences" by SPPS, native chemical ligation (NCL) and follow-up protocols.

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“…Here, we describe Click-Assisted NCL (CAN), which meets all these criteria by combining a traceless “helping hand” Lys linker (Ddap) with the bioorthogonal strained-click pair dibenzocyclooctyne (DBCO) and azide (Figure B). In the CAN strategy, peptide segments are first coupled with a Ddap linker on a Lys side chain on-resin during SPPS before functionalization with either DBCO or azide.…”

Section: Introductionmentioning

confidence: 99%

“…(2) Bioorthogonal chemistry can be performed in typical peptide-solubilizing denaturants (e.g., 6 M guanidine); (3) Bioorthogonal linkers are stable to commonly encountered CPS conditions (e.g., resin cleavage, ligation, and desulfurization); (4) Removal of bioorthogonal linkers is chemoselective, traceless, and efficient; and (5) Flexible positioning of bioorthogonal linkers within peptide sequences (e.g., placement not restricted to termini or infrequent amino acids). Here, we describe Click-Assisted NCL (CAN), which meets all these criteria by combining a traceless "helping hand" Lys linker (Ddap) 74 with the bioorthogonal strained-click pair dibenzocyclooctyne (DBCO) and azide (Figure 1B). In the CAN strategy, peptide segments are first coupled with a Ddap linker on a Lys side chain on-resin during SPPS before functionalization with either DBCO or azide.…”

Section: ■ Introductionmentioning

confidence: 99%

“…The resultant triazole linkage effectively templates the C-terminal thioester and N-terminal thiol for ligation, allowing NCL to proceed with high efficiency. After ligation, mild hydroxylamine treatment chemoselectively removes the templating linkers, 74 affording the native ligated peptide. Using model peptides, we show that the CAN strategy is compatible with all canonical amino acids.…”

Section: ■ Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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Chemical synthesis of Shiga toxin subunit B using a next-generation traceless “helping hand” solubilizing tag

Abstract: Application of a next-generation semipermanent solubilizing tag linker (Ddap) in the chemical synthesis of Shiga toxin subunit B (StxB).

Cited by 15 publications

References 50 publications

STxB as an Antigen Delivery Tool for Mucosal Vaccination

STxB as an Antigen Delivery Tool for Mucosal Vaccination

Challenges and Perspectives in Chemical Synthesis of Highly Hydrophobic Peptides

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

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