Biotechnology Letters

, Volume 40, Issue 4, pp 659–666 | Cite as

Purification of virus-like particles (VLPs) expressed in the silkworm Bombyx mori

  • Robert Minkner
  • Enoch Y. Park


Virus-like particles (VLPs) are a promising and developing option for vaccination and gene therapy. They are also interesting as shuttles for drug targeting. Currently, several different gene expression systems are available, among which the silkworm expression system is known for its mass production capacity. However, cost-effective purification with high purity of the target protein is a particular bottleneck for this system. The present review evaluates the advances in the purification of VLPs, especially from silkworm larval hemolymph. Beginning with applicable pre-treatments for VLPs over to chromatography methods and quality control of the purified VLPs. Whereupon the main focus is on the different chromatography approaches for the purification, but the structure of the VLPs and their intended use for humans make also the quality control important. Within this, the stability of the VLPs which has to be considered for the purification is as well discussed.


Chromatography Hemolymph Purification Silkworm Virus-like particle 



The authors wish to thank Dr. Tatsuya Kato of Research Institute of Green Science and Technology in Shizuoka University for critical reading of the manuscript.

Supporting Information

Supplementary Table S1—Some purifications from proteins from the silkworm or purifications from viruses and virus-like particles (VLPs) from other expression systems.

Supplementary Fig. S1—The heparin affinity purification.

Supplementary material

10529_2018_2516_MOESM1_ESM.docx (232 kb)
Supplementary material 1 (DOCX 232 kb)


  1. Catrice EVB, Sainsbury F (2015) Assembly and purification of polyomavirus-like particles from plants. Mol Biotechnol 57:904–913CrossRefPubMedGoogle Scholar
  2. Deo VK, Tsuji Y, Yasuda T, Kato T, Sakamoto N, Suzuki H, Park EY (2011) Expression of an RSV-gag virus-like particle in insect cell lines and silkworm larvae. J Virol Methods 177:147–152CrossRefPubMedGoogle Scholar
  3. Deo VK, Yoshimatsu K, Otsuki T, Dong J, Kato T, Park EY (2013) Display of Neospora caninum surface protein related sequence 2 on Rous sarcoma virus-derived gag protein virus-like particles. J Biotechnol 165:69–75CrossRefPubMedGoogle Scholar
  4. Deo VK, Kato T, Park EY (2015) Chimeric virus-like particles made using GAG and M1 capsid proteins providing dual drug delivery and vaccination platform. Mol Pharm 12:839–845CrossRefPubMedGoogle Scholar
  5. Deo VK, Kato T, Park EY (2016) Virus-like particles displaying recombinant short-chain fragment region and interleukin 2 for targeting colon cancer tumors and attracting macrophages. J Pharm Sci 105:1614–1622CrossRefPubMedGoogle Scholar
  6. Dong J, Harada M, Yoshida S, Kato Y, Murakawa A, Ogata M, Kato T, Usui T, Park EY (2013) Expression and purification of bioactive hemagglutinin protein of highly pathogenic avian influenza A (H5N1) in silkworm larvae. J Virol Methods 194:271–276CrossRefPubMedGoogle Scholar
  7. Du P, Sun S, Dong J, Zhi X, Chang Y, Teng Z, Guo H, Liu Z (2017) Purification of foot-and-mouth disease virus by heparin as ligand for certain strains. J Chromatogr B Analyt Technol Biomed Life Sci 1049–1050:16–23CrossRefPubMedGoogle Scholar
  8. Galaway FA, Stockley PG (2013) MS2 viruslike particles: a robust, semisynthetic targeted drug delivery platform. Mol Pharm 10:59–68CrossRefPubMedGoogle Scholar
  9. Geng X, Doitsh G, Yang Z, Galloway NLK, Greene WC (2014) Efficient delivery of lentiviral vectors into resting human CD4 T cells. Gene Ther 21:444–449CrossRefPubMedPubMedCentralGoogle Scholar
  10. Hahne T, Palaniyandi M, Kato T, Fleischmann P, Wätzig H, Park EY (2014) Characterization of human papillomavirus 6b L1 virus-like particles isolated from silkworms using capillary zone electrophoresis. J Biosci Bioeng 118:311–314CrossRefPubMedGoogle Scholar
  11. Hu L, Trefethen JM, Zeng Y, Yee L et al (2011) Biophysical characterization and conformational stability of Ebola and Marburg virus-like particles. J Pharm Sci 100:5156–5173CrossRefPubMedGoogle Scholar
  12. Kato T, Kajikawa M, Maenaka K, Park EY (2010) Silkworm expression system as a platform technology in life science. Appl Microbiol Biotechnol 85:459–470CrossRefPubMedGoogle Scholar
  13. Kato T, Yui M, Deo VK, Park EY (2015) Development of rous sarcoma virus-like particles displaying hcc49 scfv for specific targeted drug delivery to human colon carcinoma cells. Pharm Res 32:3699–3707CrossRefPubMedGoogle Scholar
  14. Krammer F, Schinko T, Palmberger D, Tauer C, Messner P, Grabherr R (2010) Trichoplusia ni cells (high five) are highly efficient for the production of influenza A virus-like particles: a comparison of two insect cell lines as production platforms for influenza vaccines. Mol Biotechnol 45:226–234CrossRefPubMedPubMedCentralGoogle Scholar
  15. Kröber T, Knöchlein A, Eisold K, Kalbfuß-Zimmermann B, Reichl U (2010) DNA depletion by precipitation in the purification of cell culture-derived influenza vaccines. Chem Eng Technol 33:941–959CrossRefGoogle Scholar
  16. Kumar M, Saravanan P, Jalali SK (2016) Expression and purification of virus like particles (VLPs) of foot-and-mouth disease virus in Eri silkworm (Samia cynthia ricini) larvae. Virusdisease 27:84–90CrossRefPubMedGoogle Scholar
  17. Kurosawa Y, Saito M, Kobayashi S, Okuyama T (2012) Purification of dengue virus particles by one-step ceramic hydroxyapatite chromatography. WJV 02:155–160CrossRefGoogle Scholar
  18. Lee MFX, Chan ES, Tey BT (2014) Negative chromatography: progress, applications and future perspectives. Process Biochem 49:1005–1011CrossRefGoogle Scholar
  19. Lee MFX, Chan ES, Tan WS, Tam KC, Tey BT (2015) Negative chromatography purification of hepatitis B virus-like particles using poly(oligo(ethylene glycol) methacrylate) grafted cationic adsorbent. J Chromatogr A 1415:161–165CrossRefPubMedGoogle Scholar
  20. Lee MFX, Chan ES, Tan WS, Tam KC, Tey BT (2016) Negative chromatography of hepatitis B virus-like particle: comparative study of different adsorbent designs. J Chromatogr A 1445:1–9CrossRefPubMedGoogle Scholar
  21. Leuchs B, Frehtman V, Riese M, Muller M, Rommelaere J (2017) A novel scalable, robust downstream process for oncolytic rat parvovirus: isoelectric point-based elimination of empty particles. Appl Microbiol Biotechnol 101:3143–3152CrossRefPubMedPubMedCentralGoogle Scholar
  22. Lin S-Y, Chiu H-Y, Chiang B-L, Hu Y-C (2015) Development of EV71 virus-like particle purification processes. Vaccine 33:5966–5973CrossRefPubMedGoogle Scholar
  23. Lua LHL, Connors NK, Sainsbury F, Chuan YP, Wibowo N, Middelberg APJ (2014) Bioengineering virus-like particles as vaccines. Biotechnol Bioeng 111:425–440CrossRefPubMedGoogle Scholar
  24. Marichal-Gallardo P, Pieler MM, Wolff MW, Reichl U (2017) Steric exclusion chromatography for purification of cell culture-derived influenza a virus using regenerated cellulose membranes and polyethylene glycol. J Chromatogr A 1483:110–119CrossRefPubMedGoogle Scholar
  25. Mitsudome T, Xu J, Nagata Y, Masuda A, Iiyama K, Morokuma D, Li Z, Mon H, Lee JM, Kusakabe T (2014) Expression, purification, and characterization of endo-beta-N-acetylglucosaminidase H using baculovirus-mediated silkworm protein expression system. Appl Biochem Biotechnol 172:3978–3988CrossRefPubMedGoogle Scholar
  26. Molinari P, Peralta A, Taboga O (2008) Production of rotavirus-like particles in Spodoptera frugiperda larvae. J Virol Methods 147:364–367CrossRefPubMedGoogle Scholar
  27. Morokuma D, Xu J, Hino M, Mon H, Merzaban JS, Takahashi M, Kusakabe T, Lee JM (2017) Expression and characterization of human beta-1, 4-galactosyltransferase 1 (beta4GalT1) using silkworm-baculovirus expression system. Mol Biotechnol 59:151–158CrossRefPubMedGoogle Scholar
  28. Mundle ST, Kishko M, Groppo R, DiNapoli J, Hamberger J, McNeil B, Kleanthous H, Parrington M, Zhang L, Anderson SF (2016) Core bead chromatography for preparation of highly pure, infectious respiratory syncytial virus in the negative purification mode. Vaccine 34:3690–3696CrossRefPubMedGoogle Scholar
  29. Nerome K, Matsuda S, Maegawa K, Sugita S, Kuroda K, Kawasaki K, Nerome R (2017) Quantitative analysis of the yield of avian H7 influenza virus haemagglutinin protein produced in silkworm pupae with the use of the codon-optimized DNA: a possible oral vaccine. Vaccine 35:738–746CrossRefPubMedGoogle Scholar
  30. Nestola P, Peixoto C, Silva RRJS, Alves PM, Mota JPB, Carrondo MJT (2015) Improved virus purification processes for vaccines and gene therapy. Biotechnol Bioeng 112:843–857CrossRefPubMedGoogle Scholar
  31. Palaniyandi M, Kato T, Park EY (2012) Expression of human papillomavirus 6b L1 protein in silkworm larvae and enhanced green fluorescent protein displaying on its virus-like particles. Springerplus 1:29CrossRefPubMedPubMedCentralGoogle Scholar
  32. Steppert P, Burgstaller D, Klausberger M, Berger E et al (2016) Purification of HIV-1 gag virus-like particles and separation of other extracellular particles. J Chromatogr A 1455:93–101CrossRefPubMedGoogle Scholar
  33. Steppert P, Burgstaller D, Klausberger M, Kramberger P, Tover A, Berger E, Nobauer K, Razzazi-Fazeli E, Jungbauer A (2017) Separation of HIV-1 gag virus-like particles from vesicular particles impurities by hydroxyl-functionalized monoliths. J Sep Sci 40:979–990CrossRefPubMedGoogle Scholar
  34. Strods A, Ose V, Bogans J, Cielens I, Kalnins G, Radovica I, Kazaks A, Pumpens P, Renhofa R (2015) Preparation by alkaline treatment and detailed characterisation of empty hepatitis B virus core particles for vaccine and gene therapy applications. Sci Rep 5:11639CrossRefPubMedPubMedCentralGoogle Scholar
  35. Sviben D, Forcic D, Ivancic-Jelecki J, Halassy B, Brgles M (2017) Recovery of infective virus particles in ion-exchange and hydrophobic interaction monolith chromatography is influenced by particle charge and total-to-infective particle ratio. J Chromatogr B Analyt Technol Biomed Life Sci 1054:10–19CrossRefPubMedGoogle Scholar
  36. Xue L, Liu J, Wang Q, Zhang C, Xu L, Luo J, Wang J, Qin C, Liu Y, Su Z (2017) Purification and assembling a fused capsid protein as an enterovirus 71 vaccine candidate from inclusion bodies to pentamer-based nanoparticles. Biochem Eng J 117:139–146CrossRefGoogle Scholar
  37. Yao L, Wang S, Su S, Yao N, He J, Peng L, Sun J (2012) Construction of a baculovirus-silkworm multigene expression system and its application on producing virus-like particles. PLoS ONE 7:e32510CrossRefPubMedPubMedCentralGoogle Scholar
  38. Zeltins A (2013) Construction and characterization of virus-like particles: a review. Mol Biotechnol 53:92–107CrossRefPubMedGoogle Scholar
  39. Zhao Q, Modis Y, High K, Towne V et al (2012) Disassembly and reassembly of human papillomavirus virus-like particles produces more virion-like antibody reactivity. Virol J 9:52CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Bioscience, Graduate School of Science and TechnologyShizuoka UniversityShizuokaJapan
  2. 2.Laboratory of Biotechnology, Green Chemistry Research DivisionResearch Institute of Green Science and Technology, Shizuoka UniversityShizuokaJapan

Personalised recommendations