Biosynthesis of a Fully Functional Cyclotide inside Living Bacterial Cells

Camarero, J A; Kimura, R H; Woo, Y; Cantor, J; Shekhtman, A

doi:10.1002/cbic.200700183

Title: Biosynthesis of a Fully Functional Cyclotide inside Living Bacterial Cells

Journal Article · Thu Apr 05 00:00:00 EDT 2007 · ChemBiochem, vol. 8, no. 12, August 13, 2007, pp. 1363-1366

DOI:https://doi.org/10.1002/cbic.200700183· OSTI ID:940891

Camarero, J A; Kimura, R H; Woo, Y; Cantor, J; Shekhtman, A

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.

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Research Organization:: Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

Sponsoring Organization:: USDOE

DOE Contract Number:: W-7405-ENG-48

OSTI ID:: 940891

Report Number(s):: UCRL-JRNL-230072; TRN: US200824%%387

Journal Information:: ChemBiochem, vol. 8, no. 12, August 13, 2007, pp. 1363-1366, Vol. 8, Issue 12

Country of Publication:: United States

Language:: English

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Title: Biosynthesis of a Fully Functional Cyclotide inside Living Bacterial Cells

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