Skip to main content
SpringerLink
Log in

Soluble expression, purification and functional characterization of a coil peptide composed of a positively charged and hydrophobic motif

  • Original Article
  • Published:
Amino Acids Aims and scope Submit manuscript

Abstract

A de novo heterodimeric coiled-coil system formed by the association of two synthetic peptides, the Ecoil and Kcoil, has been previously designed and proven to be an excellent and versatile tool for various biotechnology applications. However, based on the challenges encountered during its chemical synthesis, the Kcoil peptide has been designated as a “difficult peptide”. In this study, we explore the expression of the Kcoil peptide by a bacterial system as well as its subsequent purification. The maximum expression level was observed when the peptide was fused to thioredoxin and the optimized purification process consisted of three chromatographic steps: immobilized-metal affinity chromatography followed by cation-exchange chromatography and, finally, a reverse-phase high-performance liquid chromatography. This entire process led to a final volumetric production yield of 1.5 mg of pure Kcoil peptide per liter of bacterial culture, which represents a significant step towards the cost-effective production and application of coiled-coil motifs. Our results thus demonstrate for the first time that bacterial production is a viable alternative to the chemical synthesis of de novo designed coil peptides.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Aronsson C, Dånmark S, Zhou F, Öberg P, Enander K, Su H, Aili D (2015) Self-sorting heterodimeric coiled coil peptides with defined and tuneable self-assembly properties. Sci Rep 5:14063. doi:10.1038/srep14063

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Assal Y, Mizuguchi Y, Mie M, Kobatake E (2015) Growth factor tethering to protein nanoparticles via coiled-coil formation for targeted drug delivery. Bioconjug Chem 26(8):1672–1677. doi:10.1021/acs.bioconjchem.5b00266

    Article  CAS  PubMed  Google Scholar 

  • Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10(5):411–421

    Article  CAS  PubMed  Google Scholar 

  • Boucher C, St-Laurent G, Loignon M, Jolicoeur M, De Crescenzo G, Durocher Y (2008) The bioactivity and receptor affinity of recombinant tagged EGF designed for tissue engineering applications is defined by the nature and position of the tags. Tissue Eng Part A 14(12):2069–2077. doi:10.1089/ten.tea.2008.0037

    Article  CAS  PubMed  Google Scholar 

  • Boucher C, St-Laurent G, Jolicoeur M, Crescenzo GD, Durocher Y (2010) Protein detection by Western blot via coiled-coil interactions. Anal Biochem 399(1):138–140. doi:10.1016/j.ab.2009.12.007

    Article  CAS  PubMed  Google Scholar 

  • Bromley EH, Channon KJ (2011) Alpha-helical peptide assemblies giving new function to designed structures. Prog Mol Biol Transl Sci 103:231–275. doi:10.1016/B978-0-12-415906-8.00001-7

    Article  CAS  PubMed  Google Scholar 

  • Burkhard P, Stetefeld J, Strelkov SV (2001) Coiled coils: a highly versatile protein folding motif. Trends Cell Biol 11(2):82–88

    Article  CAS  PubMed  Google Scholar 

  • Chao H, Houston ME Jr, Grothe S, Kay CM, O’Connor-McCourt M, Irvin RT, Hodges RS (1996) Kinetic study on the formation of a de novo designed heterodimeric coiled-coil: use of surface plasmon resonance to monitor the association and dissociation of polypeptide chains. Biochemistry 35(37):12175–12185. doi:10.1021/bi9530604

    Article  CAS  PubMed  Google Scholar 

  • Chao H, Bautista DL, Litowski J, Irvin RT, Hodges RS (1998) Use of a heterodimeric coiled-coil system for biosensor application and affinity purification. J Chromatogr B Biomed Sci Appl 715(1):307–329

    Article  CAS  PubMed  Google Scholar 

  • Cohen C, Parry DAD (1986) α-Helical coiled coils—a widespread motif in proteins. Trends Biochem Sci 11(6):245–248. doi:10.1016/0968-0004(86)90186-6

    Article  CAS  Google Scholar 

  • Davis GD, Elisee C, Newham DM, Harrison RG (1999) New fusion protein systems designed to give soluble expression in Escherichia coli. Biotechnol Bioeng 65(4):382–388

    Article  CAS  PubMed  Google Scholar 

  • De Crescenzo G, Litowski JR, Hodges RS, O’Connor-McCourt MD (2003a) Real-time monitoring of the interactions of two-stranded de novo designed coiled-coils: effect of chain length on the kinetic and thermodynamic constants of binding. Biochemistry 42(6):1754–1763. doi:10.1021/bi0268450

    Article  PubMed  Google Scholar 

  • De Crescenzo G, Pham PL, Durocher Y, O’Connor-McCourt MD (2003b) Transforming growth factor-beta (TGF-beta) binding to the extracellular domain of the type II TGF-beta receptor: receptor capture on a biosensor surface using a new coiled-coil capture system demonstrates that avidity contributes significantly to high affinity binding. J Mol Biol 328(5):1173–1183

    Article  PubMed  Google Scholar 

  • De Crescenzo G, Pham PL, Durocher Y, Chao H, O’Connor-McCourt MD (2004) Enhancement of the antagonistic potency of transforming growth factor-beta receptor extracellular domains by coiled coil-induced homo- and heterodimerization. J Biol Chem 279(25):26013–26018. doi:10.1074/jbc.M400655200

    Article  PubMed  Google Scholar 

  • Drogoz A, David L, Rochas C, Domard A, Delair T (2007) Polyelectrolyte Complexes from Polysaccharides: formation and Stoichiometry Monitoring. Langmuir 23(22):10950–10958. doi:10.1021/la7008545

    Article  CAS  PubMed  Google Scholar 

  • Esposito D, Chatterjee DK (2006) Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol 17(4):353–358. doi:10.1016/j.copbio.2006.06.003

    Article  CAS  PubMed  Google Scholar 

  • Fortier C, De Crescenzo G, Durocher Y (2013) A versatile coiled-coil tethering system for the oriented display of ligands on nanocarriers for targeted gene delivery. Biomaterials 34(4):1344–1353. doi:10.1016/j.biomaterials.2012.10.047

    Article  CAS  PubMed  Google Scholar 

  • Gerling-Driessen UIM, Mujkic-Ninnemann N, Ponader D, Schöne D, Hartmann L, Koksch B, Gerling-Driessen UIM, Schöne D, Koksch B, Ponader D, Mujkic-Ninnemann N, Hartmann L (2015) Exploiting Oligo(amido amine) backbones for the multivalent presentation of coiled-coil peptides. Biomacromolecules 16(8):2394–2402. doi:10.1021/acs.biomac.5b00634

    Article  CAS  PubMed  Google Scholar 

  • Grigoryan G, Keating AE (2008) Structural specificity in coiled-coil interactions. Curr Opin Struct Biol 18(4):477–483. doi:10.1016/j.sbi.2008.04.008

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Guzmán F, Barberis S, Illanes A (2007) Peptide synthesis: chemical or enzymatic. Electron J Biotechnol 10(2):279–300. doi:10.2225/vol10-issue2-fulltext-13

    Article  Google Scholar 

  • Hartmann BM, Kaar W, Falconer RJ, Zeng B, Middelberg AP (2008) Expression and purification of a nanostructure-forming peptide. J Biotechnol 135(1):85–91. doi:10.1016/j.jbiotec.2008.03.003

    Article  CAS  PubMed  Google Scholar 

  • Hartmann BM, Kaar W, Yoo IK, Lua LH, Falconer RJ, Middelberg AP (2009) The chromatography-free release, isolation and purification of recombinant peptide for fibril self-assembly. Biotechnol Bioeng 104(5):973–985. doi:10.1002/bit.22447

    Article  CAS  PubMed  Google Scholar 

  • Jing XL, Luo XG, Tian WJ, Lv LH, Jiang Y, Wang N, Zhang TC (2010) High-level expression of the antimicrobial peptide plectasin in Escherichia coli. Curr Microbiol 61(3):197–202. doi:10.1007/s00284-010-9596-3

    Article  CAS  PubMed  Google Scholar 

  • Kohn WD, Hodges RS (1998) de novo design of alpha-helical coiled coils and bundles: models for the development of protein-design principles. Trends Biotechnol 16(9):379–389. doi:10.1016/S0167-7799(98)01212-8

    Article  CAS  Google Scholar 

  • Kyle S, Aggeli A, Ingham E, McPherson MJ (2010) Recombinant self-assembling peptides as biomaterials for tissue engineering. Biomaterials 31(36):9395–9405. doi:10.1016/j.biomaterials.2010.08.051

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • LaVallie ER, DiBlasio EA, Kovacic S, Grant KL, Schendel PF, McCoy JM (1993) A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnology (NY) 11(2):187–193

  • LaVallie ER, Lu Z, Diblasio-Smith EA, Collins-Racie LA, McCoy JM (2000) Thioredoxin as a fusion partner for production of soluble recombinant proteins in Escherichia coli. Methods Enzymol 326:322–340

    Article  CAS  PubMed  Google Scholar 

  • Le PU, Lenferink AE, Pinard M, Baardsnes J, Massie B, O’Connor-McCourt MD (2009) Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications. Protein Expr Purif 64(2):108–117. doi:10.1016/j.pep.2008.11.005

    Article  CAS  PubMed  Google Scholar 

  • Lee JY, Yoon CS, Chung IY, Lee YS, Lee EK (2000) Scale-up process for expression and renaturation of recombinant human epidermal growth factor from Escherichia coli inclusion bodies. Biotechnol Appl Biochem 31(Pt 3):245–248

    Article  CAS  PubMed  Google Scholar 

  • Li Y (2009) Carrier proteins for fusion expression of antimicrobial peptides in Escherichia coli. Biotechnol Appl Biochem 54(1):1–9. doi:10.1042/ba20090087

    Article  CAS  PubMed  Google Scholar 

  • Li Y (2011) Recombinant production of antimicrobial peptides in Escherichia coli: a review. Protein Expr Purif 80(2):260–267. doi:10.1016/j.pep.2011.08.001

    Article  CAS  PubMed  Google Scholar 

  • Li Y (2012) A novel protocol for the production of recombinant LL-37 expressed as a thioredoxin fusion protein. Protein Expr Purif 81(2):201–210. doi:10.1016/j.pep.2011.10.011

    Article  CAS  PubMed  Google Scholar 

  • Liberelle B, Bartholin L, Boucher C, Murschel F, Jolicoeur M, Durocher Y, Merzouki A, De Crescenzo G (2010a) New ELISA approach based on coiled-coil interactions. J Immunol Methods 362(1–2):161–167. doi:10.1016/j.jim.2010.09.027

    Article  CAS  PubMed  Google Scholar 

  • Liberelle B, Boucher C, Chen J, Jolicoeur M, Durocher Y, De Crescenzo G (2010b) Impact of epidermal growth factor tethering strategy on cellular response. Bioconjug Chem 21(12):2257–2266. doi:10.1021/bc1002604

    Article  CAS  PubMed  Google Scholar 

  • Lupas A (1996) Coiled coils: new structures and new functions. Trends Biochem Sci 21(10):375–382

    Article  CAS  PubMed  Google Scholar 

  • McCoy J, La Ville E (2001) Expression and purification of thioredoxin fusion proteins. In: Ploegh HL (ed) Current protocols in protein science, vol 28. Wiley, pp 16.8.11–16.8.14. doi:10.1002/0471140864.ps0607s10

  • Murschel F, Liberelle B, St-Laurent G, Jolicoeur M, Durocher Y, De Crescenzo G (2013) Coiled-coil-mediated grafting of bioactive vascular endothelial growth factor. Acta Biomater 9(6):6806–6813. doi:10.1016/j.actbio.2013.02.032

    Article  CAS  PubMed  Google Scholar 

  • Murschel F, Zaimi A, Noel S, Jolicoeur M, De Crescenzo G (2015) Specific adsorption via peptide tags: oriented grafting and release of growth factors for tissue engineering. Biomacromolecules. doi:10.1021/acs.biomac.5b00955

    PubMed  Google Scholar 

  • Pal G, Srivastava S (2014) Cloning and heterologous expression of plnE, -F, -J and -K genes derived from soil metagenome and purification of active plantaricin peptides. Appl Microbiol Biotechnol 98(3):1441–1447. doi:10.1007/s00253-013-5097-1

    Article  CAS  PubMed  Google Scholar 

  • Prakash A, Parsons SJ, Kyle S, McPherson MJ (2012) Recombinant production of self-assembling beta-structured peptides using SUMO as a fusion partner. Microb Cell Fact 11:92. doi:10.1186/1475-2859-11-92

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Riley JM, Aggeli A, Koopmans RJ, McPherson MJ (2009) Bioproduction and characterization of a pH responsive self-assembling peptide. Biotechnol Bioeng 103(2):241–251. doi:10.1002/bit.22274

    Article  CAS  PubMed  Google Scholar 

  • Sahdev S, Khattar SK, Saini KS (2008) Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem 307(1–2):249–264. doi:10.1007/s11010-007-9603-6

    CAS  PubMed  Google Scholar 

  • Shlyapnikov YM, Andreev YA, Kozlov SA, Vassilevski AA, Grishin EV (2008) Bacterial production of latarcin 2a, a potent antimicrobial peptide from spider venom. Protein Expr Purif 60(1):89–95. doi:10.1016/j.pep.2008.03.011

    Article  CAS  PubMed  Google Scholar 

  • Song J, Chen W, Lu Z, Hu X, Ding Y (2011) Soluble expression, purification, and characterization of recombinant human flotillin-2 (reggie-1) in Escherichia coli. Mol Biol Rep 38(3):2091–2098. doi:10.1007/s11033-010-0335-4

    Article  CAS  PubMed  Google Scholar 

  • Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60(5):523–533. doi:10.1007/s00253-002-1158-6

    Article  CAS  PubMed  Google Scholar 

  • Winkler DF, Tian K (2015) Investigation of the automated solid-phase synthesis of a 38mer peptide with difficult sequence pattern under different synthesis strategies. Amino Acids 47(4):787–794. doi:10.1007/s00726-014-1909-6

    Article  CAS  PubMed  Google Scholar 

  • Woolfson DN, Mahmoud ZN (2010) More than just bare scaffolds: towards multi-component and decorated fibrous biomaterials. Chem Soc Rev 39(9):3464–3479. doi:10.1039/c0cs00032a

    Article  CAS  PubMed  Google Scholar 

  • Xu X, Jin F, Yu X, Ji S, Wang J, Cheng H, Wang C, Zhang W (2007) Expression and purification of a recombinant antibacterial peptide, cecropin, from Escherichia coli. Protein Expr Purif 53(2):293–301. doi:10.1016/j.pep.2006.12.020

    Article  CAS  PubMed  Google Scholar 

  • Yang YL, Tian ZG, Teng D, Zhang J, Wang JR, Wang JH (2009) High-level production of a candidacidal peptide lactoferrampin in Escherichia coli by fusion expression. J Biotechnol 139(4):326–331. doi:10.1016/j.jbiotec.2009.01.003

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We would like to acknowledge the regional Center for Mass Spectrometry at University of Montreal for the peptide exact mass analysis and July Dorion-Thibaudeau and Frederic Murschel for their help during SPR assay development. This work was partly supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant program and the Canada Research Chair on Protein-enhanced Biomaterials.

Author information

Authors and Affiliations

  1. Department of Chemical Engineering, Groupe de Recherche en Sciences et Technologies Biomédicales (GRSTB), Bio-P² Research Unit, École Polytechnique de Montréal, P.O. Box 6079, Succ. Centre-Ville, Montreal, QC, H3C 3A7, Canada

    Nesrine Riahi, Olivier Henry & Gregory De Crescenzo

  2. Department of Biochemistry, Université de Montréal, PO Box 6128, Succ. Centre-Ville, Montreal, QC, H3C 3J7, Canada

    Laurent Cappadocia & James Omichinski

Authors
  1. Nesrine Riahi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Laurent Cappadocia
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Olivier Henry
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. James Omichinski
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gregory De Crescenzo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gregory De Crescenzo.

Ethics declarations

Conflict of interest

None.

Additional information

Handling Editor: J. D. Wade.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Riahi, N., Cappadocia, L., Henry, O. et al. Soluble expression, purification and functional characterization of a coil peptide composed of a positively charged and hydrophobic motif. Amino Acids 48, 567–577 (2016). https://doi.org/10.1007/s00726-015-2113-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00726-015-2113-z

Keywords

Search

Navigation