Production of a toxic polypeptide as a fusion inside GroEL cavity

The system is developed for efficient biosynthetic production of difficult-to-express polypeptides. A target polypeptide is produced fused into T. thermophilus GroEL chaperonin polypeptide chain in such a way that it is presented inside the GroEL cavity near the substrate binding surface. Such prese...

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Bibliographic Details
Published in:Scientific reports 2020-12, Vol.10 (1), p.21024, Article 21024
Main Authors: Yurkova, Maria S., Sadykhov, Elchin G., Fedorov, Alexey N.
Format: Article
Language:English
Subjects:
631/45
631/61
Amino acids
Antimicrobial Cationic Peptides - genetics
Antimicrobial Cationic Peptides - metabolism
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Chaperonin 60 - genetics
Chaperonin 60 - metabolism
Chemical treatment
Humanities and Social Sciences
Hydrophobicity
Industrial Microbiology - methods
Methionine
multidisciplinary
Peptides
Polypeptides
Protein Engineering - methods
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Science
Science (multidisciplinary)
Thermal stability
Thermus thermophilus - enzymology
Thermus thermophilus - genetics
Thermus thermophilus - metabolism
Toxicity
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Summary:The system is developed for efficient biosynthetic production of difficult-to-express polypeptides. A target polypeptide is produced fused into T. thermophilus GroEL chaperonin polypeptide chain in such a way that it is presented inside the GroEL cavity near the substrate binding surface. Such presentation allows alleviating potential problems of instability, toxicity or hydrophobicity of the fused peptide. Thermostability of thermophilic GroEL can be used for its one-step separation from the host cell proteins by heating. The target polypeptide may be released by any of amino acid-specific chemical treatments. In this study, GroEL was adapted for methionine-specific cleavage with cyanogen bromide by total replacement of methionine residues to facilitate further purification of the target polypeptide. The procedure is simple, robust and easy to scale-up. The capacity of this system to produce difficult-to-express polypeptides is demonstrated by production in bacterial system of one of the most potent antibacterial peptides polyphemusin I.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-020-78094-8