Recombinant Expression of Cyclotides Using Split Inteins

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2016 Oct 8

PMID 27714609

Citations 14

Authors

Krishnappa Jagadish

Julio A Camarero

Affiliations

Soon will be listed here.

Abstract

Cyclotides are fascinating microproteins (≈30 residues long) present in several families of plants that share a unique head-to-tail circular knotted topology of three disulfide bridges, with one disulfide penetrating through a macrocycle formed by the two other disulfides and inter-connecting peptide backbones, forming what is called a cystine knot topology. Naturally occurring cyclotides have shown to posses various pharmacologically relevant activities and have been reported to cross cell membranes. Altogether, these features make the cyclotide scaffold an excellent molecular framework for the design of novel peptide-based therapeutics, making them ideal substrates for molecular grafting of biological peptide epitopes. In this chapter we describe how to express a native folded cyclotide using intein-mediated protein trans-splicing in live Escherichia coli cells.

Citing Articles

Transgenic Innovation: Harnessing Cyclotides as Next Generation Pesticides.

Deegala S, Rathnapala H, Rajendran S, Hettiarachchi C ACS Omega. 2025; 10(7):6323-6336.

PMID: 40028067 PMC: 11865984. DOI: 10.1021/acsomega.4c09668.

Small and Versatile Cyclotides as Anti-infective Agents.

Candido E, Gasparetto L, Luchi L, Pimentel J, Cardoso M, Macedo M ACS Infect Dis. 2025; 11(2):386-397.

PMID: 39842000 PMC: 11833872. DOI: 10.1021/acsinfecdis.4c00957.

Efficient splicing of the CPE intein derived from directed evolution of the PRP8 intein.

Zhan Q, Shi C, Jiang Y, Gao X, Lin Y Acta Biochim Biophys Sin (Shanghai). 2023; 55(8):1310-1318.

PMID: 37489009 PMC: 10448054. DOI: 10.3724/abbs.2023135.

Lipidation of a bioactive cyclotide-based CXCR4 antagonist greatly improves its pharmacokinetic profile in vivo.

Chaudhuri D, Lu T, Jacob B, Abraham S, Shankar P, Poss M J Control Release. 2023; 359:26-32.

PMID: 37236320 PMC: 10527528. DOI: 10.1016/j.jconrel.2023.05.026.

Using the Cyclotide Scaffold for Targeting Biomolecular Interactions in Drug Development.

Jacob B, Vogelaar A, Cadenas E, Camarero J Molecules. 2022; 27(19).

PMID: 36234971 PMC: 9570680. DOI: 10.3390/molecules27196430.

References

Austin J, Wang W, Puttamadappa S, Shekhtman A, Camarero J . Biosynthesis and biological screening of a genetically encoded library based on the cyclotide MCoTI-I. Chembiochem. 2009; 10(16):2663-70. PMC: 2940853. DOI: 10.1002/cbic.200900534. View

Chan L, Gunasekera S, Henriques S, Worth N, Le S, Clark R . Engineering pro-angiogenic peptides using stable, disulfide-rich cyclic scaffolds. Blood. 2011; 118(25):6709-17. DOI: 10.1182/blood-2011-06-359141. View

Camarero J, Kimura R, Woo Y, Shekhtman A, Cantor J . Biosynthesis of a fully functional cyclotide inside living bacterial cells. Chembiochem. 2007; 8(12):1363-6. DOI: 10.1002/cbic.200700183. View

Hernandez J, Gagnon J, Chiche L, Nguyen T, Andrieu J, Heitz A . Squash trypsin inhibitors from Momordica cochinchinensis exhibit an atypical macrocyclic structure. Biochemistry. 2000; 39(19):5722-30. DOI: 10.1021/bi9929756. View

Gould A, Ji Y, Aboye T, Camarero J . Cyclotides, a novel ultrastable polypeptide scaffold for drug discovery. Curr Pharm Des. 2011; 17(38):4294-307. PMC: 3330703. DOI: 10.2174/138161211798999438. View

Aboye T, Ha H, Majumder S, Christ F, Debyser Z, Shekhtman A . Design of a novel cyclotide-based CXCR4 antagonist with anti-human immunodeficiency virus (HIV)-1 activity. J Med Chem. 2012; 55(23):10729-34. PMC: 3521869. DOI: 10.1021/jm301468k. View

Huang Y, Colgrave M, Clark R, Kotze A, Craik D . Lysine-scanning mutagenesis reveals an amendable face of the cyclotide kalata B1 for the optimization of nematocidal activity. J Biol Chem. 2010; 285(14):10797-805. PMC: 2856286. DOI: 10.1074/jbc.M109.089854. View

Austin J, Kimura R, Woo Y, Camarero J . In vivo biosynthesis of an Ala-scan library based on the cyclic peptide SFTI-1. Amino Acids. 2009; 38(5):1313-22. PMC: 2936557. DOI: 10.1007/s00726-009-0338-4. View

Simonsen S, Sando L, Rosengren K, Wang C, Colgrave M, Daly N . Alanine scanning mutagenesis of the prototypic cyclotide reveals a cluster of residues essential for bioactivity. J Biol Chem. 2008; 283(15):9805-13. DOI: 10.1074/jbc.M709303200. View

10.

Kimura R, Tran A, Camarero J . Biosynthesis of the cyclotide Kalata B1 by using protein splicing. Angew Chem Int Ed Engl. 2005; 45(6):973-6. DOI: 10.1002/anie.200503882. View

11.

Zettler J, Schutz V, Mootz H . The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS Lett. 2009; 583(5):909-14. DOI: 10.1016/j.febslet.2009.02.003. View

12.

Ji Y, Majumder S, Millard M, Borra R, Bi T, Elnagar A . In vivo activation of the p53 tumor suppressor pathway by an engineered cyclotide. J Am Chem Soc. 2013; 135(31):11623-11633. PMC: 3767463. DOI: 10.1021/ja405108p. View

13.

Jagadish K, Gould A, Borra R, Majumder S, Mushtaq Z, Shekhtman A . Recombinant Expression and Phenotypic Screening of a Bioactive Cyclotide Against α-Synuclein-Induced Cytotoxicity in Baker's Yeast. Angew Chem Int Ed Engl. 2015; 54(29):8390-4. PMC: 4545262. DOI: 10.1002/anie.201501186. View

14.

Sancheti H, Camarero J . "Splicing up" drug discovery. Cell-based expression and screening of genetically-encoded libraries of backbone-cyclized polypeptides. Adv Drug Deliv Rev. 2009; 61(11):908-17. PMC: 3329944. DOI: 10.1016/j.addr.2009.07.003. View

15.

Cascales L, Henriques S, Kerr M, Huang Y, Sweet M, Daly N . Identification and characterization of a new family of cell-penetrating peptides: cyclic cell-penetrating peptides. J Biol Chem. 2011; 286(42):36932-43. PMC: 3196123. DOI: 10.1074/jbc.M111.264424. View

16.

Mylne J, Chan L, Chanson A, Daly N, Schaefer H, Bailey T . Cyclic peptides arising by evolutionary parallelism via asparaginyl-endopeptidase-mediated biosynthesis. Plant Cell. 2012; 24(7):2765-78. PMC: 3426113. DOI: 10.1105/tpc.112.099085. View

17.

Daly N, Rosengren K, Craik D . Discovery, structure and biological activities of cyclotides. Adv Drug Deliv Rev. 2009; 61(11):918-30. DOI: 10.1016/j.addr.2009.05.003. View

18.

Contreras J, Elnagar A, Hamm-Alvarez S, Camarero J . Cellular uptake of cyclotide MCoTI-I follows multiple endocytic pathways. J Control Release. 2011; 155(2):134-43. PMC: 3195939. DOI: 10.1016/j.jconrel.2011.08.030. View

19.

Daly N, Thorstholm L, Greenwood K, King G, Rosengren K, Heras B . Structural insights into the role of the cyclic backbone in a squash trypsin inhibitor. J Biol Chem. 2013; 288(50):36141-8. PMC: 3861661. DOI: 10.1074/jbc.M113.528240. View

20.

Saether O, Craik D, Campbell I, Sletten K, JUUL J, Norman D . Elucidation of the primary and three-dimensional structure of the uterotonic polypeptide kalata B1. Biochemistry. 1995; 34(13):4147-58. DOI: 10.1021/bi00013a002. View