ArticleNature protocols2021
Yeast-based bioproduction of disulfide-rich peptides and their cyclization via asparaginyl endopeptidases.
Article in Nature protocols, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 17 papers.
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Who cites it
17 citing papers in PubMed, 41 citations in OpenAlex.
- Site-specific protein and peptide modification by redeploying an asparaginyl ligase for noncanonical reactions.Nature protocols · 2026Review
- Tangled Tail of Mechanically Interlocked Peptides.Journal of the American Chemical Society · 2026Review
- Butyric Acid-Modified m-P14 Peptide Ameliorates Anti-Glomerular Basement Membrane Disease.International journal of molecular sciences · 2026Article
- Advances in cyclotide research: bioactivity to cyclotide-based therapeutics.Molecular diversity · 2025Review
- Asparaginyl Ligases with Engineered Substrate Specificity for Controlled, Sequential Transpeptidation Reactions.Journal of the American Chemical Society · 2025Article
- Cyclization of the Analgesic α-Conotoxin Vc1.1 With a Non-Natural Linker: Effects on Structure, Stability, and Bioactivity.Journal of peptide science : an official publication of the European Peptide Society · 2025Article
- Exploring Use of a Protein Cage System for Producing Bioactive Peptides in Escherichia coli.Microbial biotechnology · 2025Article
- Identification of a key peptide cyclase for novel cyclic peptide discovery in Pseudostellaria heterophylla.Plant communications · 2025Article
- Yeast as a tool for exploring disulfide-rich peptides.FEMS yeast research · 2025Review
- Repurposing a plant peptide cyclase for targeted lysine acylation.Nature chemistry · 2024Article
- Insights into the synthesis strategies of plant-derived cyclotides.Amino acids · 2023Review
- Native and Engineered Cyclic Disulfide-Rich Peptides as Drug Leads.Molecules (Basel, Switzerland) · 2023Review
- Peptide/protein-based macrocycles: from biological synthesis to biomedical applications.RSC chemical biology · 2022Review
- Cystine Knot Peptides with Tuneable Activity and Mechanism.Angewandte Chemie (International ed. in English) · 2022Article
- Enzymatic C-to-C Protein Ligation.Angewandte Chemie (International ed. in English) · 2022Article
- Evaluation of Efficient Non-reducing Enzymatic and Chemical Ligation Strategies for Complex Disulfide-Rich Peptides.Bioconjugate chemistry · 2021Article
- Plant derived cyclic peptides.Biochemical Society transactions · 2021Review
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Authors and funding
11 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Cyclic disulfide-rich peptides have attracted significant interest in drug development and biotechnology. Here, we describe a protocol for producing cyclic peptide precursors in Pichia pastoris that undergo in vitro enzymatic maturation into cyclic peptides using recombinant asparaginyl endopeptidases (AEPs). Peptide precursors are expressed with a C-terminal His tag and secreted into the media, enabling facile purification by immobilized metal affinity chromatography. After AEP-mediated cyclization, cyclic peptides are purified by reverse-phase high-performance liquid chromatography and characterized by mass spectrometry, peptide mass fingerprinting, NMR spectroscopy, and activity assays. We demonstrate the broad applicability of this protocol by generating cyclic peptides from three distinct classes that are either naturally occurring or synthetically backbone cyclized, and range in size from 14 amino acids with one disulfide bond, to 34 amino acids with a cystine knot comprising three disulfide bonds. The protocol requires 14 d to identify and optimize a high-expressing Pichia clone in small-scale cultures (24 well plates or 50 mL tubes), after which large-scale production in a bioreactor and peptide purification can be completed in 10 d. We use the cyclotide Momordica cochinchinensis trypsin inhibitor II as an example. We also include a protocol for recombinant AEP production in Escherichia coli as AEPs are emerging tools for orthogonal peptide and protein ligation. We focus on two AEPs that preferentially cyclize different peptide precursors, namely an engineered AEP with improved catalytic efficiency [C247A]OaAEP1
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