Abstract
Cyclization of the peptide backbone by connecting the N- and C-terminus can endow target peptides with favorable properties, such as increased stability or potential oral bioavailability. However, there are few tools available for carrying out this modification. Asparaginyl endopeptidases (AEPs) are a class of enzymes that typically work as proteases, but a subset is highly efficient at cyclization of the peptide backbone. In this chapter we describe how to utilize a cyclizing AEP (OaAEP1b) to produce backbone-cyclized peptides both in planta and in vitro. Using the in planta method, OaAEP1b and the target precursor peptide are coexpressed in the leaves of the model plant Nicotiana benthamiana, and cyclization of the target peptide occurs in planta. Using the in vitro method, purified recombinant OaAEP1b produced in bacteria is used to cyclize the target precursor peptide in vitro.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Clark RJ, Jensen J, Nevin ST et al (2010) The engineering of an orally active conotoxin for the treatment of neuropathic pain. Angew Chem Int Ed Engl 49:6545–6548
Chan LY, Zhang VM, Huang Y et al (2013) Cyclization of the antimicrobial peptide gomesin with native chemical ligation: influences on stability and bioactivity. ChemBioChem 14:617–624
Wong CTT, Rowlands DK, Wong CH et al (2012) Orally active peptidic bradykinin B 1 receptor antagonists engineered from a cyclotide scaffold for inflammatory pain treatment. Angew Chem Int Ed 51:5620–5624
Shafee T, Harris K, Anderson M (2015) Biosynthesis of cyclotides. In: Craik DJ (ed) Advances in botanical research, plant cyclotides. Elsevier Ltd, London
Harris KS, Durek T, Kaas Q et al (2015) Efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase. Nat Commun 6:10199
Nguyen GKT, Wang S, Qiu Y et al (2014) Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nat Chem Biol 10:732–738
Jackson MA, Gilding EK, Shafee T et al (2018) Molecular basis for the production of cyclic peptides by plant asparaginyl endopeptidases. Nat Commun 9:2411
Poon S, Harris KS, Jackson MA et al (2018) Co-expression of a cyclizing asparaginyl endopeptidase enables efficient production of cyclic peptides in planta. J Exp Bot 69:633–641
Nguyen GKT, Cao Y, Wang W et al (2015) Site-specific N-terminal labeling of peptides and proteins using butelase 1 and thiodepsipeptide. Angew Chem Int Ed 54:15694–15698
Cao Y, Nguyen GKT, Chuah S et al (2016) Butelase-mediated ligation as an efficient bioconjugation wethod for the synthesis of peptide dendrimers. Bioconjug Chem 27:2592–2596
Craik DJ, Daly NL, Bond T et al (1999) Plant cyclotides: a unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif. J Mol Biol 294:1327–1336
Colgrave ML, Craik DJ (2004) Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: the importance of the cyclic cystine knot. Biochemistry 43:5965–5975
Jennings C, West J, Waine C et al (2001) Biosynthesis and insecticidal properties of plant cyclotides : the cyclic knotted proteins from Oldenlandia affinis. Proc Natl Acad Sci U S A 98:10614–10619
Plan MR, Saska I, Cagauan AG et al (2008) Backbone cyclised peptides from plants show molluscicidal activity against the rice pest Pomacea canaliculata (Golden Apple Snail). J Agric Food Chem 56:5237–5241
Colgrave ML, Kotze AC, Huang Y et al (2008) Cyclotides: natural, circular plant peptides that possess significant activity against gastrointestinal nematode parasites of Sheep. Biochem 47:5581–5589
Colgrave ML, Kotze AC, Kopp S et al (2009) Anthelmintic activity of cyclotides: in vitro studies with canine and human hookworms. Acta Trop 109:163–166
Poth AG, Chan LY, Craik DJ (2013) Cyclotides as grafting frameworks for protein engineering and drug design applications. Biopolymers 100:480–491
Sparkes IA, Runions J, Kearns A et al (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 1:2019–2025
Harris KS, Casey JL, Coley AM et al (2005) Binding hot spot for invasion inhibitory molecules on Plasmodium falciparum apical membrane antigen 1. Infect Immun 73:6981–6989
Catanzariti A, Soboleva TA, Jans DA et al (2004) An efficient system for high-level expression and easy purification of authentic recombinant proteins. Protein Sci 13:1331–1339
Peyret H, Lomonossoff GP (2013) The pEAQ vector series: the easy and quick way to produce recombinant proteins in plants. Plant Mol Biol 83:51–58
Gleba YY, Tusé D, Giritch A (2013) Plant viral vectors for delivery by Agrobacterium. In: Palmer K, Gleba Y (eds) Plant viral vectors. Current topics in microbiology and immunology. Springer, Berlin
Horsch RB, Fry JE, Hoffmann NL et al (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231
Yeshak MY, Burman R, Eriksson C et al (2012) Optimization of cyclotide extraction parameters. Phytochem Lett 5:776–781
Craik DJ, Henriques ST, Mylne JS et al (2012) Cyclotide isolation and characterization. Methods Enzymol 516:37–62
Copeland RA (2002) Enzymes: a practical introduction to structure, mechanism, and data analysis, 2nd edn. Wiley-VCH, Inc, New York, NY
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Harris, K.S., Poon, S., Quimbar, P., Anderson, M.A. (2019). In Vitro and In Planta Cyclization of Target Peptides Using an Asparaginyl Endopeptidase from Oldenlandia affinis. In: Nuijens, T., Schmidt, M. (eds) Enzyme-Mediated Ligation Methods. Methods in Molecular Biology, vol 2012. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9546-2_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9546-2_12
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9545-5
Online ISBN: 978-1-4939-9546-2
eBook Packages: Springer Protocols