skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: 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}
}

Works referenced in this record:

The cyclotide family of circular miniproteins: Nature's combinatorial peptide template
journal, January 2006


Biosynthesis of the Cyclotide Kalata B1 by Using Protein Splicing
journal, January 2006


Semisynthesis of Proteins by Expressed Protein Ligation
journal, June 2003


Retrovirally Delivered Random Cyclic Peptide Libraries Yield Inhibitors of Interleukin-4 Signaling in Human B Cells
journal, July 2002


Circulins A and B. Novel human immunodeficiency virus (HIV)-inhibitory macrocyclic peptides from the tropical tree Chassalia parvifolia.
journal, October 1994


Circular Proteins in Plants: SOLUTION STRUCTURE OF A NOVEL MACROCYCLIC TRYPSIN INHIBITOR FROM MOMORDICA COCHINCHINENSIS
journal, April 2001


Plant cyclotides: A unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif
journal, December 1999


Squash Inhibitors: From Structural Motifs to Macrocyclic Knottins
journal, October 2004


Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid.
journal, November 1989


An unusual structural motif of antimicrobial peptides containing end-to-end macrocycle and cystine-knot disulfides
journal, August 1999


Crystal structure of GyrA intein from Mycobacterium xenopi reveals structural basis of protein splicing
journal, January 1998


Dissecting the Chemistry of Protein Splicing and Its Applications
journal, February 2000


Biosynthesis of the Cyclotide Kalata B1 by Using Protein Splicing
journal, January 2006


Conserved Structural and Sequence Elements Implicated in the Processing of Gene-encoded Circular Proteins
journal, August 2004


Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis
journal, September 2001


Twists, Knots, and Rings in Proteins: STRUCTURAL DEFINITION OF THE CYCLOTIDE FRAMEWORK
journal, December 2002


Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm
journal, November 1999


Squash Trypsin Inhibitors from Momordica cochinchinensis Exhibit an Atypical Macrocyclic Structure
journal, May 2000


Solution Structure of the Squash Trypsin Inhibitor MCoTI-II. A New Family for Cyclic Knottins ,
journal, July 2001


Cyclopsychotride A, a Biologically Active, 31-Residue Cyclic Peptide Isolated from Psychotria longipes
journal, December 1994


Biosynthesis of a Head-to-Tail Cyclized Protein with Improved Biological Activity
journal, June 1999


Intracellular protein interaction mapping with FRET hybrids
journal, November 2006


Chemical Synthesis of a Circular Protein Domain: Evidence for Folding-Assisted Cyclization
journal, February 1998


Synthesis of Native Proteins by Chemical Ligation
journal, June 2000


Protein-Spleißen: Mechanismus und Anwendungen
journal, February 2000


Structural plasticity of the cyclic-cystine-knot framework: implications for biological activity and drug design
journal, January 2006


Trypsin inhibition by macrocyclic and open-chain variants of the squash inhibitor MCoTI-II
journal, January 2005