Biosynthesis of a Fully Functional Cyclotide inside Living Bacterial Cells
Abstract
The cyclotide MCoTI-II is a powerful trypsin inhibitor recently isolated from the seeds of Momordica cochinchinensis, a plant member of cucurbitaceae family. We report for the first time the in vivo biosynthesis of natively-folded MCoTI-II inside live E. coli cells. Our biomimetic approach involves the intracellular backbone cyclization of a linear cyclotide-intein fusion precursor mediated by a modified protein splicing domain. The cyclized peptide then spontaneously folds into its native conformation. The use of genetically engineered E. coli cells containing mutations in the glutathione and thioredoxin reductase genes considerably improves the production of folded MCoTI-II in vivo. Biochemical and structural characterization of the recombinant MCoTI-II confirmed its identity. Biosynthetic access to correctly-folded cyclotides allows the possibility of generating cell-based combinatorial libraries that can be screened inside living cells for their ability to modulate or inhibit cellular processes.
- Authors:
- Publication Date:
- Research Org.:
- Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 940891
- Report Number(s):
- UCRL-JRNL-230072
TRN: US200824%%387
- DOE Contract Number:
- W-7405-ENG-48
- Resource Type:
- Journal Article
- Journal Name:
- ChemBiochem, vol. 8, no. 12, August 13, 2007, pp. 1363-1366
- Additional Journal Information:
- Journal Volume: 8; Journal Issue: 12
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; BIOSYNTHESIS; CYCLIZATION; FUNCTIONALS; GENES; GLUTATHIONE; IN VIVO; MUTATIONS; OXIDOREDUCTASES; PEPTIDES; PRECURSOR; PRODUCTION; PROTEINS; SEEDS; SPLICING; TRYPSIN
Citation Formats
Camarero, J A, Kimura, R H, Woo, Y, Cantor, J, and Shekhtman, A. Biosynthesis of a Fully Functional Cyclotide inside Living Bacterial Cells. United States: N. p., 2007.
Web. doi:10.1002/cbic.200700183.
Camarero, J A, Kimura, R H, Woo, Y, Cantor, J, & Shekhtman, A. Biosynthesis of a Fully Functional Cyclotide inside Living Bacterial Cells. United States. https://doi.org/10.1002/cbic.200700183
Camarero, J A, Kimura, R H, Woo, Y, Cantor, J, and Shekhtman, A. 2007.
"Biosynthesis of a Fully Functional Cyclotide inside Living Bacterial Cells". United States. https://doi.org/10.1002/cbic.200700183. https://www.osti.gov/servlets/purl/940891.
@article{osti_940891,
title = {Biosynthesis of a Fully Functional Cyclotide inside Living Bacterial Cells},
author = {Camarero, J A and Kimura, R H and Woo, Y and Cantor, J and Shekhtman, A},
abstractNote = {The cyclotide MCoTI-II is a powerful trypsin inhibitor recently isolated from the seeds of Momordica cochinchinensis, a plant member of cucurbitaceae family. We report for the first time the in vivo biosynthesis of natively-folded MCoTI-II inside live E. coli cells. Our biomimetic approach involves the intracellular backbone cyclization of a linear cyclotide-intein fusion precursor mediated by a modified protein splicing domain. The cyclized peptide then spontaneously folds into its native conformation. The use of genetically engineered E. coli cells containing mutations in the glutathione and thioredoxin reductase genes considerably improves the production of folded MCoTI-II in vivo. Biochemical and structural characterization of the recombinant MCoTI-II confirmed its identity. Biosynthetic access to correctly-folded cyclotides allows the possibility of generating cell-based combinatorial libraries that can be screened inside living cells for their ability to modulate or inhibit cellular processes.},
doi = {10.1002/cbic.200700183},
url = {https://www.osti.gov/biblio/940891},
journal = {ChemBiochem, vol. 8, no. 12, August 13, 2007, pp. 1363-1366},
number = 12,
volume = 8,
place = {United States},
year = {Thu Apr 05 00:00:00 EDT 2007},
month = {Thu Apr 05 00:00:00 EDT 2007}
}
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