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Recombinant Protein Production in Yeast pp 309–322Cite as

Guidelines for Small-Scale Production and Purification of Hepatitis B Surface Antigen Virus-Like Particles from Recombinant Pichia pastoris

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1923))

Abstract

Virus-like particle (VLP)-based vaccines have been in the market since decades for preventing viral infection and have proven their usefulness also in other areas of biotechnology. Here, we describe in detail simple small-scale production and purification procedures for the generation of hepatitis B surface antigen (HBsAg) VLPs using Pichia pastoris as expression host. This protocol may also be applicable with variations to other HBsAg-based VLPs additionally carrying antigens of other pathogens.

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References

  1. Cregg JM, Tschopp JF, Stillman C, Siegel R, Akong M, Craig WS et al (1987) High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris. Nat Biotechnol 5:479–485

    Article  CAS  Google Scholar 

  2. Kushnir N, Streatfield SJ, Yusibov V (2012) Virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development. Vaccine 31:58–83

    Article  CAS  Google Scholar 

  3. Boigard H, Alimova A, Martin GR, Katz A, Gottlieb P, Galarza JM (2017) Zika virus-like particle (VLP) based vaccine. PLoS Negl Trop Dis 11:e0005608

    Article  Google Scholar 

  4. Meireles LC, Marinho RT, Van Damme P (2015) Three decades of hepatitis B control with vaccination. World J Hepatol 7:2127–2132

    Article  Google Scholar 

  5. Roldao A, Mellado MC, Castilho LR, Carrondo MJ, Alves PM (2010) Virus-like particles in vaccine development. Expert Rev Vaccines 9:1149–1176

    Article  CAS  Google Scholar 

  6. Zhao Q, Li S, Yu H, Xia N, Modis Y (2013) Virus-like particle-based human vaccines: quality assessment based on structural and functional properties. Trends Biotechnol 31:654–663

    Article  Google Scholar 

  7. Khetarpal N, Poddar A, Nemani SK, Dhar N, Patil A, Negi P et al (2013) Dengue-specific subviral nanoparticles: design, creation and characterization. J Nanobiotechnology 11:15

    Article  CAS  Google Scholar 

  8. Nardin EH, Oliveira GA, Calvo-Calle JM, Wetzel K, Maier C, Birkett AJ et al (2004) Phase I testing of a malaria vaccine composed of hepatitis B virus core particles expressing Plasmodium falciparum circumsporozoite epitopes. Infect Immun 72:6519–6527

    Article  CAS  Google Scholar 

  9. Zhang L, Lua LH, Middelberg AP, Sun Y, Connors NK (2015) Biomolecular engineering of virus-like particles aided by computational chemistry methods. Chem Soc Rev 44:8608–8618

    Article  CAS  Google Scholar 

  10. Kim HJ, Kim HJ (2017) Yeast as an expression system for producing virus-like particles: what factors do we need to consider? Lett Appl Microbiol 64:111–123

    Article  CAS  Google Scholar 

  11. Wampler DE, Lehman ED, Boger J, McAleer WJ, Scolnick EM (1985) Multiple chemical forms of hepatitis B surface antigen produced in yeast. Proc Natl Acad Sci U S A 82:6830–6834

    Article  CAS  Google Scholar 

  12. Biemans R, Thines D, Petre-Parent B, De Wilde M, Rutgers T, Cabezon T (1992) Immunoelectron microscopic detection of the hepatitis B virus major surface protein in dilated perinuclear membranes of yeast cells. DNA Cell Biol 11:621–626

    Article  CAS  Google Scholar 

  13. Gurramkonda C, Adnan A, Gabel T, Lünsdorf H, Ross A, Nemani SK et al (2009) Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: application to intracellular production of hepatitis B surface antigen. Microb Cell Factories 8:13

    Article  Google Scholar 

  14. Vanz AL, Lünsdorf H, Adnan A, Nimtz M, Gurramkonda C, Khanna N et al (2012) Physiological response of Pichia pastoris GS115 to methanol-induced high level production of the hepatitis B surface antigen: catabolic adaptation, stress responses, and autophagic processes. Microb Cell Factories 11:103

    Article  CAS  Google Scholar 

  15. Gurramkonda C, Zahid M, Nemani SK, Adnan A, Gudi SK, Khanna N et al (2013) Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties. J Chromatogr B 940:104–111

    Article  CAS  Google Scholar 

  16. Zahid M, Lünsdorf H, Rinas U (2015) Assessing stability and assembly of the hepatitis B surface antigen into virus-like particles during down-stream processing. Vaccine 33:3739–3745

    Article  CAS  Google Scholar 

  17. Vassileva A, Chugh DA, Swaminathan S, Khanna N (2001) Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris. Protein Expr Purif 21:71–80

    Article  CAS  Google Scholar 

  18. Bardiya N (2006) Expression in and purification of Hepatitis B surface antigen (S-protein) from methylotrophic yeast Pichia pastoris. Anaerobe 12:194–203

    Article  CAS  Google Scholar 

  19. Ottone S, Nguyen X, Bazin J, Berard C, Jimenez S, Letourneur O (2007) Expression of hepatitis B surface antigen major subtypes in Pichia pastoris and purification for in vitro diagnosis. Protein Expr Purif 56:177–188

    Article  CAS  Google Scholar 

  20. Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM, Carnemolla B et al (2004) Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25:1327–1333

    Article  CAS  Google Scholar 

  21. Kanno T, Yamada T, Iwabuki H, Tanaka H, Kuroda S, Tanizawa K et al (2002) Size distribution measurement of vesicles by atomic force microscopy. Anal Biochem 309:196–199

    Article  CAS  Google Scholar 

  22. McEvoy M, Razinkov V, Wei Z, Casas-Finet JR, Tous GI, Schenerman MA (2011) Improved particle counting and size distribution determination of aggregated virus populations by asymmetric flow field-flow fractionation and multiangle light scattering techniques. Biotechnol Prog 27:547–554

    Article  CAS  Google Scholar 

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Acknowledgments

We are grateful to C. Gurramkonda for critical comments.

Author information

Authors and Affiliations

  1. Technical Chemistry—Life Science, Leibniz University of Hannover, Hannover, Germany

    Maria Zahid & Ursula Rinas

  2. Department of Biology, Lahore University of Management Sciences, Lahore, Pakistan

    Maria Zahid

  3. Helmholtz Centre for Infection Research, Braunschweig, Germany

    Ursula Rinas

Authors
  1. Maria Zahid
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  2. Ursula Rinas
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Corresponding author

Correspondence to Ursula Rinas .

Editor information

Editors and Affiliations

  1. Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU) and Austrian Centre of Industrial Biotechnology (acib), Vienna, Austria

    Brigitte Gasser

  2. Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU) and Austrian Centre of Industrial Biotechnology (acib), Vienna, Austria

    Diethard Mattanovich

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Zahid, M., Rinas, U. (2019). Guidelines for Small-Scale Production and Purification of Hepatitis B Surface Antigen Virus-Like Particles from Recombinant Pichia pastoris . In: Gasser, B., Mattanovich, D. (eds) Recombinant Protein Production in Yeast. Methods in Molecular Biology, vol 1923. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9024-5_14

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  • DOI: https://doi.org/10.1007/978-1-4939-9024-5_14

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9023-8

  • Online ISBN: 978-1-4939-9024-5

  • eBook Packages: Springer Protocols

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