Synthesis of cholera toxin B subunit glycoconjugates using site-specific orthogonal oxime and sortase ligation reactions

Dolan, Jonathan P.; Machin, Darren C.; Dedola, Simone; Field, Robert A.; Webb, Michael E.; Turnbull, W. Bruce

doi:10.3389/fchem.2022.958272

“…We next investigated modification of the C‐terminus of the pentameric cholera toxin B‐subunit (CTB) which is widely used as a tool in cell biology and neuroscience [20] . We have previously reported a CTB construct with a C‐terminal sortase recognition site, however periplasmic expression yielded heterogeneous substrates in which only 80–90 % of the protomer chains included the desired extension [21] . We therefore generated a new C‐terminally His‐tagged CTB construct incorporating a LPETGA labelling motif.…”

Section: Resultsmentioning

confidence: 99%

“…[20] We have previously reported a CTB construct with a C-terminal sortase recognition site, however periplasmic expression yielded heterogeneous substrates in which only 80-90 % of the protomer chains included the desired extension. [21] We therefore generated a new C-terminally His-tagged CTB construct incorporating a LPETGA labelling motif. Cytosolic expression of the unfolded protein into inclusion bodies, followed by dissolution in 8 M urea, affinity purification subsequent refolding provided homogeneous material for the labelling experiments.…”

Section: Methodsmentioning

confidence: 99%

Quantitative N‐ or C‐Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d‐Aminopeptidase

Arnott,

Morgan,

Hollingsworth

et al. 2024

Angew Chem Int Ed

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Quantitative and selective labelling of proteins is widely used in both academic and industrial laboratories, and catalytic labelling of proteins using transpeptidases such as sortases has proved to be a popular strategy for selective modification. A major challenge for this class of enzymes is that the majority of procedures require an excess of the labelling reagent or, alternatively, activated substrates rather than simple commercially‐sourced peptides. We report the use of a coupled enzyme strategy which enables quantitative N‐ and C‐terminal labelling of proteins using un‐activated labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence‐specific degradation of the peptide by‐product, shifting the equilibriums to favor product formation which greatly enhances the reaction efficiency. Subsequent optimization of the reaction allows N‑terminal labelling of proteins using essentially equimolar ratios of peptide label to protein and C‐terminal labelling with only a small excess. Minimizing the amount of substrate required for quantitative labelling has the potential to improve industrial processes and facilitate the use of transpeptidation as a method for protein labelling.

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“…[20] We have previously reported a CTB construct with a C-terminal sortase recognition site, however periplasmic expression yielded heterogeneous substrates in which only 80-90 % of the protomer chains included the desired extension. [21] We therefore generated a new C-terminally His-tagged CTB construct incorporating a LPETGA labelling motif. Cytosolic expression of the unfolded protein into inclusion bodies, followed by dissolution in 8 M urea, affinity purification and subsequent refolding provided homogeneous material for the labelling experiments.…”

Section: Optimisation Of C-terminal Labellingmentioning

confidence: 99%

Quantitative N‐ or C‐Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d‐Aminopeptidase

Arnott,

Morgan,

Hollingsworth

et al. 2024

Angewandte Chemie

Self Cite

0

View full text Add to dashboard Cite

Quantitative and selective labelling of proteins is widely used in both academic and industrial laboratories, and catalytic labelling of proteins using transpeptidases such as sortases has proved to be a popular strategy for selective modification. A major challenge for this class of enzymes is that the majority of procedures require an excess of the labelling reagent or, alternatively, activated substrates rather than simple commercially‐sourced peptides. We report the use of a coupled enzyme strategy which enables quantitative N‐ and C‐terminal labelling of proteins using un‐activated labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence‐specific degradation of the peptide by‐product, shifting the equilibriums to favor product formation which greatly enhances the reaction efficiency. Subsequent optimization of the reaction allows N‑terminal labelling of proteins using essentially equimolar ratios of peptide label to protein and C‐terminal labelling with only a small excess. Minimizing the amount of substrate required for quantitative labelling has the potential to improve industrial processes and facilitate the use of transpeptidation as a method for protein labelling.

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“…CTB is most often used as an adjuvant for immunological studies [ 9 ], or for trafficking cargoes into cell interiors, such as fluorophores [ 10 ], nanoparticles [ 6 ], biologics [ 11 ], and antigens [ 12 ]. CTB is a structurally robust protein, making it resistant to modification, thus CTB has already been used in a number of bioengineering and synthetic biology pursuits, such as assembly into higher order geometries [ 13 ], mediating membrane fusion [ 14 ], and labeled via sortase ligation [ 15 ]. The cholera toxin (CT) ( Figure 1 A) trafficking pathway initiates when CTB binds to glycans presented on the cell surface [ 16 , 17 , 18 ], including ganglioside GM1 (shown in black in Figure 1 C), triggering endocytosis [ 19 ] and retrograde trafficking.…”

Section: Introductionmentioning

confidence: 99%

The Mutagenic Plasticity of the Cholera Toxin B-Subunit Surface Residues: Stability and Affinity

Au,

Manfield,

Webb

et al. 2024

Toxins

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Mastering selective molecule trafficking across human cell membranes poses a formidable challenge in healthcare biotechnology while offering the prospect of breakthroughs in drug delivery, gene therapy, and diagnostic imaging. The cholera toxin B-subunit (CTB) has the potential to be a useful cargo transporter for these applications. CTB is a robust protein that is amenable to reengineering for diverse applications; however, protein redesign has mostly focused on modifications of the N- and C-termini of the protein. Exploiting the full power of rational redesign requires a detailed understanding of the contributions of the surface residues to protein stability and binding activity. Here, we employed Rosetta-based computational saturation scans on 58 surface residues of CTB, including the GM1 binding site, to analyze both ligand-bound and ligand-free structures to decipher mutational effects on protein stability and GM1 affinity. Complimentary experimental results from differential scanning fluorimetry and isothermal titration calorimetry provided melting temperatures and GM1 binding affinities for 40 alanine mutants among these positions. The results showed that CTB can accommodate diverse mutations while maintaining its stability and ligand binding affinity. These mutations could potentially allow modification of the oligosaccharide binding specificity to change its cellular targeting, alter the B-subunit intracellular routing, or impact its shelf-life and in vivo half-life through changes to protein stability. We anticipate that the mutational space maps presented here will serve as a cornerstone for future CTB redesigns, paving the way for the development of innovative biotechnological tools.

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Synthesis of cholera toxin B subunit glycoconjugates using site-specific orthogonal oxime and sortase ligation reactions

Cited by 4 publications

References 67 publications

Quantitative N‐ or C‐Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d‐Aminopeptidase

Quantitative N‐ or C‐Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d‐Aminopeptidase

Quantitative N‐ or C‐Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d‐Aminopeptidase

The Mutagenic Plasticity of the Cholera Toxin B-Subunit Surface Residues: Stability and Affinity

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