Recombinant Expression of Cyclotides Using Expressed Protein Ligation

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2020 Mar 8

PMID 32144675

Citations 3

Authors

Maria Jose Campbell

Jingtan Su

Julio A Camarero

Affiliations

Soon will be listed here.

Abstract

Cyclotides are naturally occurring microproteins (≈30 residues long) present in several families of plants. All cyclotides share a unique head-to-tail circular knotted topology containing three disulfide bridges forming a cystine knot topology. Cyclotides possess high stability to chemical, physical, and biological degradation and have been reported to cross cellular membranes. In addition, naturally occurring and engineered cyclotides have shown to possess various pharmacologically relevant activities. These unique features make the cyclotide scaffold an excellent tool for the design of novel peptide-based therapeutics by using molecular evolution and/or peptide epitope grafting techniques. In this chapter, we provide protocols to recombinantly produce a natively folded cyclotide making use of a standard bacterial expression system in combination with an intein-mediated backbone cyclization with concomitant oxidative folding.

Citing Articles

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.

Engineered Cyclotides with Potent Broad in Vitro and in Vivo Antimicrobial Activity.

Ganesan R, Dughbaj M, Ramirez L, Beringer S, Aboye T, Shekhtman A Chemistry. 2021; 27(49):12702-12708.

PMID: 34159664 PMC: 8410672. DOI: 10.1002/chem.202101438.

References

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

Gillon A, Saska I, Jennings C, Guarino R, Craik D, Anderson M . Biosynthesis of circular proteins in plants. Plant J. 2007; 53(3):505-15. DOI: 10.1111/j.1365-313X.2007.03357.x. View

Jagadish K, Borra R, Lacey V, Majumder S, Shekhtman A, Wang L . Expression of fluorescent cyclotides using protein trans-splicing for easy monitoring of cyclotide-protein interactions. Angew Chem Int Ed Engl. 2013; 52(11):3126-31. PMC: 3767473. DOI: 10.1002/anie.201209219. 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

Saska I, Gillon A, Hatsugai N, Dietzgen R, Hara-Nishimura I, Anderson M . An asparaginyl endopeptidase mediates in vivo protein backbone cyclization. J Biol Chem. 2007; 282(40):29721-8. DOI: 10.1074/jbc.M705185200. View

Puttamadappa S, Jagadish K, Shekhtman A, Camarero J . Backbone dynamics of cyclotide MCoTI-I free and complexed with trypsin. Angew Chem Int Ed Engl. 2010; 49(39):7030-4. PMC: 2944905. DOI: 10.1002/anie.201002906. 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

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

Klabunde T, Sharma S, Telenti A, Jacobs Jr W, Sacchettini J . Crystal structure of GyrA intein from Mycobacterium xenopi reveals structural basis of protein splicing. Nat Struct Biol. 1998; 5(1):31-6. DOI: 10.1038/nsb0198-31. 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.

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

12.

Chaudhuri D, Aboye T, Camarero J . Using backbone-cyclized Cys-rich polypeptides as molecular scaffolds to target protein-protein interactions. Biochem J. 2019; 476(1):67-83. PMC: 7080216. DOI: 10.1042/BCJ20180792. View

13.

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

14.

Bessette P, Aslund F, Beckwith J, Georgiou G . Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc Natl Acad Sci U S A. 1999; 96(24):13703-8. PMC: 24128. DOI: 10.1073/pnas.96.24.13703. View

15.

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

16.

Wong C, Rowlands D, Wong C, Lo T, Nguyen G, Li H . Orally active peptidic bradykinin B1 receptor antagonists engineered from a cyclotide scaffold for inflammatory pain treatment. Angew Chem Int Ed Engl. 2012; 51(23):5620-4. DOI: 10.1002/anie.201200984. View

17.

Jagadish K, Camarero J . Recombinant Expression of Cyclotides Using Split Inteins. Methods Mol Biol. 2016; 1495:41-55. PMC: 5117428. DOI: 10.1007/978-1-4939-6451-2_4. View

18.

Berrade L, Camarero J . Expressed protein ligation: a resourceful tool to study protein structure and function. Cell Mol Life Sci. 2009; 66(24):3909-22. PMC: 3806878. DOI: 10.1007/s00018-009-0122-3. View

19.

Gould A, Camarero J . Cyclotides: Overview and Biotechnological Applications. Chembiochem. 2017; 18(14):1350-1363. PMC: 5812342. DOI: 10.1002/cbic.201700153. View

20.

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