Development of ULYSSIS, a Tool for the Biosynthesis of Cyclotides and Cyclic Knottins

Handley, Thomas N. G.; Kleffmann, Torsten; Butler, Margaret I.

doi:10.1007/s10989-021-10336-3

Cited by 3 publications

(1 citation statement)

References 42 publications

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“…The production of His-NKC-Cys-DOPA 5 increased as the TCEP concentration increased from 0 to 10 mM; however, at 20 mM, it decreased ( Figure 4 b). The optimal TCEP concentration for maximum ligation efficiency was determined to be 10 mM, which is the concentration used for intein-mediated protein ligation in previous studies [ 40 , 41 ]; therefore, we employed a ligation buffer containing 10 mM TCEP in all further experiments. In addition, His-NKC-Cys-DOPA 5 production was higher at an HNG-to-Cys-DOPA 5 molar ratio of 1:2.5 (0.2 mM HNG and 0.5 mM Cys-DOPA 5 ) than at a ratio of 1:5 (0.2 mM HNG and 1.0 mM Cys-DOPA 5 ); hence, a higher molar ratio of Cys-DOPA 5 to HNG was not required.…”

Section: Resultsmentioning

confidence: 99%

Semi-Biosynthetic Production of Surface-Binding Adhesive Antimicrobial Peptides Using Intein-Mediated Protein Ligation

Hwang

Cho

et al. 2022

IJMS

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Microbial infections remain a global health concern, calling for the urgent need to implement effective prevention measures. Antimicrobial peptides (AMPs) have been extensively studied as potential antimicrobial coating agents. However, an efficient and economical method for AMP production is lacking. Here, we synthesized the direct coating adhesive AMP, NKC-DOPA5, composed of NKC, a potent AMP, and repeats of the adhesive amino acid 3,4-dihydroxyphenylalanine (DOPA) via an intein-mediated protein ligation strategy. NKC was expressed as a soluble fusion protein His-NKC-GyrA (HNG) in Escherichia coli, comprising an N-terminal 6× His-tag and a C-terminal Mxe GyrA intein. The HNG protein was efficiently produced in a 500-L fermenter, with a titer of 1.63 g/L. The NKC-thioester was released from the purified HNG fusion protein by thiol attack and subsequently ligated with chemically synthesized Cys-DOPA5. The ligated peptide His-NKC-Cys-DOPA5 was obtained at a yield of 88.7%. The purified His-NKC-Cys-DOPA5 possessed surface-binding and antimicrobial properties identical to those of the peptide obtained via solid-phase peptide synthesis. His-NKC-Cys-DOPA5 can be applied as a practical and functional antimicrobial coating to various materials, such as medical devices and home appliances.

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Section: Resultsmentioning

confidence: 99%

Semi-Biosynthetic Production of Surface-Binding Adhesive Antimicrobial Peptides Using Intein-Mediated Protein Ligation

Hwang

Cho

et al. 2022

IJMS

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Recombinant Production of The Cyclotide Kalata B1 by Conditional Split Inteins

Yayci,

Huang,

Harvey

et al. 2024

ChemBioChem

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This study describes the design, production, and characterization of a novel conditional intein system for the recombinant production of cyclic peptides. The system is based on two key features: (1) a promiscuous extein recognition site allowing cyclization of virtually any peptide, and (2) a secondary split site within the intein itself enabling triggered splicing at will. Two intein precursors were recombinantly expressed, purified, and then self‐assembled in vitro to cyclize the model peptide kalata B1 (kB1). Cyclized kB1 was successfully purified, refolded, and characterized by mass spectrometry and NMR, demonstrating correct disulfide bond formation and identical structure to synthetic kB1. Importantly, the intein‐derived kB1 retained full biological activity as evidenced by insect cell toxicity assays. This work establishes a versatile and efficient approach for intein‐mediated protein cyclization with potential applications in bioengineering and peptide discovery.

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Enhancing cyclotide bioproduction: harnessing biological synthesis methods and various expression systems for large-scale manufacturing

Taghizadeh,

Niazi,

Mirzapour-Kouhdasht

et al. 2024

Critical Reviews in Biotechnology

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Development of ULYSSIS, a Tool for the Biosynthesis of Cyclotides and Cyclic Knottins

Cited by 3 publications

References 42 publications

Semi-Biosynthetic Production of Surface-Binding Adhesive Antimicrobial Peptides Using Intein-Mediated Protein Ligation

Semi-Biosynthetic Production of Surface-Binding Adhesive Antimicrobial Peptides Using Intein-Mediated Protein Ligation

Recombinant Production of The Cyclotide Kalata B1 by Conditional Split Inteins

Enhancing cyclotide bioproduction: harnessing biological synthesis methods and various expression systems for large-scale manufacturing

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