Applied Microbiology and Biotechnology

, Volume 103, Issue 2, pp 833–842 | Cite as

Efficient production of porcine circovirus virus-like particles using the nonconventional yeast Kluyveromyces marxianus

  • Jinkun Duan
  • Deqiang Yang
  • Lei Chen
  • Yao Yu
  • Jungang ZhouEmail author
  • Hong LuEmail author
Applied genetics and molecular biotechnology


Porcine circovirus type 2 (PCV2) is a ubiquitous virus with high pathogenicity closely associated with the postweaning multisystemic wasting syndrome (PMWS) and porcine circovirus diseases (PCVDs), which caused significant economic losses in the swine industry worldwide every year. The PCV2 virus-like particles (VLPs) are a powerful subunit vaccine that can elicit high immune response due to its native PCV2 virus morphology. The baculovirus expression system is the widely used platform for producing commercial PCV2 VLP vaccines, but its yield and cost limited the development of low-cost vaccines for veterinary applications. Here, we applied a nonconventional yeast Kluyveromyces marxianus to enhance the production of PCV2 VLPs. After codon optimization, the PCV2 Cap protein was expressed in K. marxianus and assemble spontaneously into VLPs. Using a chemically defined medium, we achieved approximately 1.91 g/L of PCV2 VLP antigen in a 5-L bioreactor after high cell density fermentation for 72 h. That yield greatly exceeded to recently reported PCV2 VLPs obtained by baculovirus-insect cell, Escherichia coli and Pichia pastoris. By the means of two-step chromatography, 652.8 mg of PCV2 VLP antigen was obtained from 1 L of the recombinant K. marxianus cell culture. The PCV2 VLPs induced high level of anti-PCV2 IgG antibody in mice serums and decreased the virus titers in both livers and spleens of the challenged mice. These results illustrated that K. marxianus is a powerful yeast for cost-effective production of PCV2 VLP vaccines.


Porcine circovirus Capsid protein Virus-like particles Kluyveromyces marxianus 


Compliance with ethical standards


This study was supported by the projects of National Natural Science Foundation of China (31770094 and 91731310), National High Technology Research and Development Program of China (2014AA021301), Science and Technology Research Program of Shanghai (18391901800) and Open Research Funds of the State Key Laboratory of Genetic Engineering, Fudan University.

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

All the mice experimental procedures were approved by the Animal Experiment Committee of Fudan University. All applicable international, national, and institutional guidelines for the care and use of animals were strictly followed.


  1. Alegria-Schaffer A (2014) Western blotting using chemiluminescent substrates. Methods Enzymol 541:251–259Google Scholar
  2. Allan G, Krakowka S, Ellis J, Charreyre C (2012) Discovery and evolving history of two genetically related but phenotypically different viruses, porcine circoviruses 1 and 2. Virus Res 164:4–9CrossRefGoogle Scholar
  3. Antunes DF, de Souza CG, de Morais MA (2000) A simple and rapid method for lithium acetate-mediated transformation of Kluyveromyces marxianus cells. World J Microbiol Biotechnol 16:653–654CrossRefGoogle Scholar
  4. Bucarey SA, Noriega J, Reyes P, Tapia C, Saenz L, Zuniga A, Tobar JA (2009) The optimized capsid gene of porcine circovirus type 2 expressed in yeast forms virus-like particles and elicits antibody responses in mice fed with recombinant yeast extracts. Vaccine 27:5781–5790CrossRefGoogle Scholar
  5. Cai XP, Zhang J, Yuan HY, Fang ZA, Li YY (2005) Secretory expression of heterologous protein in Kluyveromyces cicerisporus. Appl Microbiol Biotechnol 67:364–369CrossRefGoogle Scholar
  6. Chae C (2005) A review of porcine circovirus 2-associated syndromes and diseases. Vet J 169:326–336CrossRefGoogle Scholar
  7. Chen Y, Zhang Y, Quan C, Luo J, Yang YL, Yu MR, Kong YJ, Ma GH, Su ZG (2015) Aggregation and antigenicity of virus like particle in salt solution-a case study with hepatitis B surface antigen. Vaccine 33:4300–4306CrossRefGoogle Scholar
  8. Cheung AK, Bolin SR (2002) Kinetics of porcine circovirus type 2 replication. Arch Virol 147:43–58CrossRefGoogle Scholar
  9. Cruz TF, Magro AJ, de Castro A, Pedraza-Ordonez FJ, Tsunemi MH, Perahia D, Araujo JP Jr (2018) In vitro and in silico studies reveal capsid-mutant porcine circovirus 2b with novel cytopathogenic and structural characteristics. Virus Res 251:22–33CrossRefGoogle Scholar
  10. Ding Y, Chuan YP, He LZ, Middelberg APJ (2010) Modeling the competition between aggregation and self-sssembly during virus-like particle processing. Biotechnol Bioeng 107:550–560CrossRefGoogle Scholar
  11. Fachinger V, Bischoff R, Ben Jedidia S, Saalmuller A, Elbers K (2008) The effect of vaccination against porcine circovirus type 2 in pigs suffering from porcine respiratory disease complex. Vaccine 26:1488–1499CrossRefGoogle Scholar
  12. Finlay BB, McFadden G (2006) Anti-immunology: evasion of the host immune system by bacterial and viral pathogens. Cell 124:767–782CrossRefGoogle Scholar
  13. Firth C, Charleston MA, Duffy S, Shapiro B, Holmes EC (2009) Insights into the evolutionary history of an emerging livestock pathogen: porcine circovirus 2. J Virol 83:12813–12821CrossRefGoogle Scholar
  14. Franzo G, Cortey M, Segales J, Hughes J, Drigo M (2016) Phylodynamic analysis of porcine circovirus type 2 reveals global waves of emerging genotypes and the circulation of recombinant forms. Mol Phylogenet Evol 100:269–280CrossRefGoogle Scholar
  15. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–U341CrossRefGoogle Scholar
  16. Gillespie J, Opriessnig T, Meng XJ, Pelzer K, Buechner-Maxwell V (2009) Porcine circovirus type 2 and porcine circovirus-associated disease. J Vet Intern Med 23:1151–1163CrossRefGoogle Scholar
  17. Groeneveld P, Stouthamer AH, Westerhoff HV (2009) Super life - how and why 'cell selection' leads to the fastest-growing eukaryote. FEBS J 276:254–270CrossRefGoogle Scholar
  18. Hamel AL, Lin LL, Nayar GPS (1998) Nucleotide sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs. J Virol 72:5262–5267Google Scholar
  19. Hensing M, Vrouwenvelder H, Hellinga C, Baartmans R, Vandijken H (1994) Production of extracellular inulinase in high-cell-density fed-batch cultures of Kluyveromyces marxianus. Appl Microbiol Biotechnol 42:516–521CrossRefGoogle Scholar
  20. Jiang CG, Wang G, Tu YB, Liu YG, Wang SJ, Cai XH, An TQ (2017) Genetic analysis of porcine circovirus type 2 in China. Arch Virol 162:2715–2726CrossRefGoogle Scholar
  21. Jin JY, Park CH, Cho SH, Chung JH (2018) The level of decoy epitope in PCV2 vaccine affects the neutralizing activity of sera in the immunized animals. Biochem Biophys Res Commun 496:846–851CrossRefGoogle Scholar
  22. Kiupel M, Stevenson GW, Choi J, Latimer KS, Kanitz CL, Mittal SK (2001) Viral replication and lesions in BALB/c mice experimentally inoculated with porcine circovirus isolated from a pig with postweaning multisystemic wasting disease. Vet Pathol 38:74–82CrossRefGoogle Scholar
  23. Liu QG, Tikoo SK, Babiuk LA (2001) Nuclear localization of the ORF2 protein encoded by porcine circovirus type 2. Virology 285:91–99CrossRefGoogle Scholar
  24. Liu J, Chen I, Kwang J (2005) Characterization of a previously unidentified viral protein in porcine circovirus type 2-infected cells and its role in virus-induced apoptosis. J Virol 79:8262–8274CrossRefGoogle Scholar
  25. Liu J, Chen I, Du QY, Chua HK, Kwang J (2006) The ORF3 protein of porcine circovirus type 2 is involved in viral pathogenesis in vivo. J Virol 80:5065–5073CrossRefGoogle Scholar
  26. Liu YK, Zhang YY, Yao LG, Hao HF, Fu XJ, Yang ZQ, Du EQ (2015) Enhanced production of porcine circovirus type 2 (PCV2) virus-like particles in Sf9 cells by translational enhancers. Biotechnol Lett 37:1765–1771CrossRefGoogle Scholar
  27. Lopez-Vidal J, Gomez-Sebastian S, Barcena J, Nunez MD, Martinez-Alonso D, Dudognon B, Guijarro E, Escribano JM (2015) Improved production efficiency of virus-like particles by the baculovirus expression vector system. PLoS One 10:e0140039CrossRefGoogle Scholar
  28. Lua LHL, Connors NK, Sainsbury F, Chuan YP, Wibowo N, Middelberg APJ (2014) Bioengineering virus-like particles as vaccines. Biotechnol Bioeng 111:425–440CrossRefGoogle Scholar
  29. Lv QZ, Guo KK, Wang T, Zhang CC, Zhang YM (2015) Porcine circovirus type 2 ORF4 protein binds heavy chain ferritin. J Biosci (Bangalore) 40:477–485CrossRefGoogle Scholar
  30. Marcekova Z, Psikal I, Kosinova E, Benada O, Sebo P, Bumba L (2009) Heterologous expression of full-length capsid protein of porcine circovirus 2 in Escherichia coli and its potential use for detection of antibodies. J Virol Methods 162:133–141CrossRefGoogle Scholar
  31. Masuda A, Lee JM, Miyata T, Sato T, Hayashi S, Hino M, Morokuma D, Karasaki N, Mon H, Kusakabe T (2018) Purification and characterization of immunogenic recombinant virus-like particles of porcine circovirus type 2 expressed in silkworm pupae. J Gen Virol 99:917–926CrossRefGoogle Scholar
  32. Misinzo G, Meerts P, Bublot M, Mast J, Weingart HM, Nauwynck HJ (2005) Binding and entry characteristics of porcine circovirus 2 in cells of the porcine monocytic line 3D4/31. J Gen Virol 86:2057–2068CrossRefGoogle Scholar
  33. Nainys J, Lasickiene R, Petraityte-Burneikiene R, Dabrisius J, Lelesius R, Sereika V, Zvirbliene A, Sasnauskas K, Gedvilaite A (2014) Generation in yeast of recombinant virus-like particles of porcine circovirus type 2 capsid protein and their use for a serologic assay and development of monoclonal antibodies. BMC Biotechnol 14:100CrossRefGoogle Scholar
  34. Nawagitgul P, Morozov I, Bolin SR, Harms FA, Sorden SD, Paul PS (2000) Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein. J Gen Virol 81:2281–2287CrossRefGoogle Scholar
  35. Opriessnig T, Xiao CT, Gerber PF, Halbur PG (2013) Emergence of a novel mutant PCV2b variant associated with clinical PCVAD in two vaccinated pig farms in the US concurrently infected with PPV2. Vet Microbiol 163:177–183CrossRefGoogle Scholar
  36. Opriessnig T, Xiao CT, Halbur PG, Gerber PF, Matzinger SR, Meng XJ (2017) A commercial porcine circovirus (PCV) type 2a-based vaccine reduces PCV2d viremia and shedding and prevents PCV2d transmission to naive pigs under experimental conditions. Vaccine 35:248–254CrossRefGoogle Scholar
  37. Phan TG, Giannitti F, Rossow S, Marthaler D, Knutson TP, Li L, Deng X, Resende T, Vannucci F, Delwart E (2016) Detection of a novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation. Virol J 13:184CrossRefGoogle Scholar
  38. Puigbo P, Guzman E, Romeu A, Garcia-Vallve S (2007) OPTIMIZER: a web server for optimizing the codon usage of DNA sequences. Nucleic Acids Res 35:W126–W131CrossRefGoogle Scholar
  39. Ren LZ, Chen XR, Ouyang HS (2016) Interactions of porcine circovirus 2 with its hosts. Virus Genes 52:437–444CrossRefGoogle Scholar
  40. Rouwenhorst RJ, Visser LE, Vanderbaan AA, Scheffers WA, Vandijken JP (1988) Production, distribution, and kinetic-properties of inulinase in continuous cultures of Kluyveromyces marxianus CBS 6556. Appl Environ Microbiol 54:1131–1137Google Scholar
  41. Shi L, Sanyal G, Ni A, Luo Z, Doshna S, Wang B, Graham TL, Wang N, Volkin DB (2005) Stabilization of human papillomavirus virus-like particles by non-ionic surfactants. J Pharm Sci 94:1538–1551CrossRefGoogle Scholar
  42. Steppert P, Burgstallera D, Klausberger M, Tover A, Berger E, Jungbauer A (2017) Quantification and characterization of virus-like particles by size-exclusion chromatography and nanoparticle tracking analysis. J Chromatogr 1487:89–99CrossRefGoogle Scholar
  43. Summers MD (2006) Milestones leading to the genetic engineering of baculoviruses as expression vector systems and viral pesticides. Adv Virus Res 68:3–73CrossRefGoogle Scholar
  44. Tischer I, Rasch R, Tochtermann G (1974) Characterization of papovavirus-and picornavirus-like particles in permanent pig kidney cell lines. Zentralbl Bakteriol Orig A 226:153–167Google Scholar
  45. Tischer I, Gelderblom H, Vettermann W, Koch MA (1982) A very small porcine virus with circular single-stranded-DNA. Nature 295:64–66CrossRefGoogle Scholar
  46. Trible BR, Suddith AW, Kerrigan MA, Cino-Ozuna AG, Hesse RA, Rowland RR (2012) Recognition of the different structural forms of the capsid protein determines the outcome following infection with porcine circovirus type 2. J Virol 86:13508–13514CrossRefGoogle Scholar
  47. Trundova M, Celer V (2007) Expression of porcine circovirus 2 ORF2 gene requires codon optimized E. coli cells. Virus Genes 34:199–204CrossRefGoogle Scholar
  48. Tu YB, Wang YQ, Wang G, Wu JN, Liu YG, Wang SJ, Jiang CG, Cai XH (2013) High-level expression and immunogenicity of a porcine circovirus type 2 capsid protein through codon optimization in Pichia pastoris. Appl Microbiol Biotechnol 97:2867–2875CrossRefGoogle Scholar
  49. Ulmer JB, Valley U, Rappuoli R (2006) Vaccine manufacturing: challenges and solutions. Nat Biotechnol 24:1377–1383CrossRefGoogle Scholar
  50. Wu PC, Lin WL, Wu CM, Chi JN, Chien MS, Huang CJ (2012) Characterization of porcine circovirus type 2 (PCV2) capsid particle assembly and its application to virus-like particle vaccine development. Appl Microbiol Biotechnol 95:1501–1507CrossRefGoogle Scholar
  51. Wu PC, Chen TY, Chi JN, Chien MS, Huang CJ (2016) Efficient expression and purification of porcine circovirus type 2 virus-like particles in Escherichia coli. J Biotechnol 220:78–85CrossRefGoogle Scholar
  52. Yang X, Chen FW, Cao YH, Pang DX, Ouyang HS, Ren LZ (2013) Comparative analysis of different methods to enhance porcine circovirus 2 replication. J Virol Methods 187:368–371CrossRefGoogle Scholar
  53. Yu C, Li X, Liu JW, Diao WZ, Zhang LC, Xiao Y, Wei HF, Yu YL, Yu YQ, Wang LY (2016) Replacing the decoy epitope of PCV2b capsid protein with a protective epitope enhances efficacy of PCV2b vaccine. Vaccine 34:6358–6366CrossRefGoogle Scholar
  54. Zaveckas M, Snipaitis S, Pesliakas H, Nainys J, Gedvilaite A (2015) Purification of recombinant virus-like particles of porcine circovirus type 2 capsid protein using ion-exchange monolith chromatography. J Chromatogr B 991:21–28CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Genetic Engineering, School of Life SciencesFudan UniversityShanghaiChina
  2. 2.Shanghai Engineering Research Center of Industrial MicroorganismsShanghaiChina

Personalised recommendations