Purification and analysis of integrity and components of VLPs Virus-like particles (VLPs) as vaccines, vectors and adjuvants Fondation Mérieux Conference Center Veyrier du Lac – France April 1, 2014 Andris Zeltiņš Latvian Biomedical Research and Study Centre, Ratsupites 1, Riga, LV-1067, Latvia [email protected] Purification and analysis of integrity and components of VLPs History: First VLPs at Latvian Biomedical Research & Study Centre HBcAg VLPs (1987) Qβ VLPs (1993) Bacteriophage fr VLPs (1987) G. Borisova, M. Bundule, E. Grinstein, D. Dreilina, A. Dreimane, J. Kalis, T. Kozlovskaya, V. Loseva, V. Ose, P. Pumpen, P. Pushko, D. Snikere, E. Stankevica, V. Tsibinogin, E.J. Gren . Mol. Gen. (Life Sci. Adv.) 1987, 6: 169· 174 Kozlovska TM, Cielens I, Dreilina D, Dislers A, Baumanis V, Ose V, Pumpens P. Gene. 1993; 137(1): 133-7. Purification and analysis of integrity and components of VLPs Examples of VLPs constructed at Latvian Biomedical research & study centre AP205 P. pastoris CB5 P. pastoris fr GA PP7 Qβ SP S. cerevisiae P. pastoris E.coli P. pastoris E.coli Bacterial VLPs ApMV RBDV CCMV E.coli E.coli E.coli RYMV (T3) E.coli RYMV(T1) E.coli RGMoV CfMV P. pastoris E.coli Plant VLPs ACLSV E.coli ASPV ASGV PVX PVY PVM E.coli E. coli E.coli E.coli E.coli HBcAg Mammalian VLPs E.coli / yeasts / mammalian cells HaPyV E.coli / Drosophila S2 Gene synthesis Hospital / veterinary specimen Environmental samples Purification and analysis of integrity and components of VLPs How to obtain new VLPs? Bacteria Yeasts Insect cells Viral nucleic acid CP cDNA cloning Mammalian cells direct CP cDNA cloning Plants Cell-free synthesis - Cell disruption Early identification Purification Components of VLPs Stability Storage VLPs-? Purification and analysis of integrity and components of VLPs Buffer systems preserving the integrity of VLPs - Ionic strength (high/low salt content); Metal ions (e.g. Ca2+, Mg 2+); Chelating aģents (e.g. EDTA); Reducing agents (e.g. DTT, mercaptoethanol); Protease inhibitors (e.g., EDTA, PMSF, protease inhibition «cocktails»); Urea; Nucleases (DNAses, RNAses, nuclease benzonase etc.); Detergents (e.g. TX-100, Tween 20); Precipitation with ammonium sulfate or PEG; - For new VLPs – buffer systems derived from isolation protocols of corresponding native viruses ensuring the structural integrity and/or infectivity; Purification and analysis of integrity and components of VLPs Cell disruption Freezing / thawing grinding with Al2O3 French press RYMV-K VLPs RGMoV VLPs E.coli P.pastoris Ultrasonication focused, non-contact RGMoV VLPs P.pastoris Ultrasonication conventional RYMV VLPs E.coli Cell disruption have to be efficient and preserve the integrity of VLPs RYMV-K VLPs? Purification and analysis of integrity and components of VLPs Procedures useful for early detection of VLPs Analytical ultracentrifugation / SDS/PAGE Typical VLPs, no structures in background M S P 1 2 3 4 5 6 No VLPs found Visual test S P M 1 2 3 4 5 6 Some VLPs found, most CP in aggregates M S P 1 2 3 4 5 6 Purification and analysis of integrity and components of VLPs Procedures useful for early detection of VLPs Agarose gels / Crude lysates M - + M A1 A2 A1 A2 - + - + - + - + Benzonase treatment VLPs A2 1 VLPs 1 2 3 1 2 - + - + - + - + Benzonase treatment 3 - M + VLPs 2 Agarose gel analysis can suggest the presence of VLPs in the samples 1 2 3 3 10 3 10 3 10 ul After PEG8000 precipitation VLPs 3? Purification and analysis of integrity and components of VLPs Purification: ultracentrifuge gradient separation M T S1 P1 S2 P2 1 2 3 4 5 6 7 ApMV VLP purification: SW32 rotor (Beckman), 25 000 rpm, 6 h, discontinuous sucrose gradient (20-60%, 1xPBS, 0.5% TX-100), fraction size 5 ml M 1 2 3 4 5 6 7 8 Second sucrose gradient (10-40%, 1xPBS, 0.5% TX-100), fraction size 4 ml 9 ApMV VLPs Purification and analysis of integrity and components of VLPs Purification: column chromatography Ion exchange chromatography Size-exclusion chromatography RYMV VLPs; Fractogel TMAE, 20 mM Tris/HCl; 0 – 2M NaCl HBc- E1 M S 8 10 12 17 19 24 28 29 34 35 40 42 49 Fract. No. RYMV VLPs Skrastina D, Petrovskis I, Petraityte R, Sominskaya I, Ose V, Lieknina I, Bogans J, Sasnauskas K, Pumpens P. Clin Vaccine Immunol. 2013, 20 (11):1719-28 HBc VLPs Purification and analysis of integrity and components of VLPs Characterization of VLPs: mass spectrometry SDS/PAGE M 0 T Western blot 1 M 0 T 1 1 - PVY CP Mw theor = 30 053 1 - PVY VLPs M GNDTIDAGGSTKKDAKQEQGSIQPNLNKEKEK DVNVGTSGTYTVPRIK... Kalnciema I, Skrastina D, Ose V, Pumpens P, Zeltins A. Mol Biotechnol. 2012; 52(2):129-39. Purification and analysis of integrity and components of VLPs Characterization of VLPs: mass spectrometry 1 2 SDS/PAGE 1 2 phosphoprotein staining 1 - HBc VLPs 2 - phosphorylated HBc VLPs E.coli P. pastoris Freivalds J, Dislers A, Ose V, Pumpens P, Tars K, Kazaks A. Protein Expr Purif. 2011; 75(2): 218-224 Purification and analysis of integrity and components of VLPs Characterization of VLPs: dynamic light scattering HBc- E1 HBc VLPs Skrastina D, Petrovskis I, Petraityte R, Sominskaya I, Ose V, Lieknina I, Bogans J, Sasnauskas K, Pumpens P. Clin Vaccine Immunol. 2013, 20(11):1719-28 RGMoV VLPs (d=28.5 nm) P. pastoris Dynamic light scattering can serve as an alternative tool for EM Purification and analysis of integrity and components of VLPs Characterization of VLPs: 3D crystallographic models Bacteriophage ϕCB5 VLPs ϕCB5 3D structural model VLP crystals VLPs can serve as an alternative source of viral structures for crystallographic studies Plevka P, Kazaks A, Voronkova T, Kotelovica S, Dishlers A, Liljas L, Tars K. J Mol Biol. 2009; 391(3) :635-47. Purification and analysis of integrity and components of VLPs Stability studies: Sypro Orange / Real-time PCR (melting point determination) Principle: - Solution containing VLPs is not activating Sypro Orange fluorescence at room temperature (hydrophobic surfaces are hidden); - Thermal denaturation expose the hydrophobic surfaces of VLPs and cause Sypro Orange binding and fluorescence; - VLP stability curve and its midpoint value (melting temperature) can be obtained by changing the temperature gradually to unfold the protein and measure the change in fluorescence . 15 mM phosphate +50 mM DTT +25 mM EDTA +0.5 M NaCl RGMoV virions 78oC 76oC 65oC 80oC RGMoV VLPs 74oC 73oC 64oC 70oC RGMoV virions RGMoV VLPs from P. pastoris Purification and analysis of integrity and components of VLPs Stability studies: storage of VLPs - Optimal VLP concentration; Salt concentration Additives (e.g., glycerol, sucrose, trehaloze, sorbitol, polysorbat); Freezing (-20oC/-80oC) / storage at +4oC Aggregated VLPs after freezing Disassembled VLPs after freezing Purification and analysis of integrity and components of VLPs Characterization of VLPs: introduced epitopes Mass spectrometry PVY VLPs PVY CP Competitive ELISA PVY-preS1 VLPs PVY-preS1 CP M GNDTIDAGGSTKKDAKQEQGSIQPNLNKEKEK DVN... M GNDNPLGFFPDHQLDPAFRANTANPGSGGGGSGGGGSGGGGS TIDAGGSTKKDAKQEQGSIQPNLNKEKEK DVN The surface localization of the preS1 epitope was demonstrated by competitive ELISA. Increasing amounts of competitor antigen (PVY‐CP‐preS1; PvyCP,) were added to the preS1‐specific mAb MA18/7 and the binding of antibodies to the immobilized preS1 peptide was measured. Kalnciema I, Skrastina D, Ose V, Pumpens P, Zeltins A. Mol Biotechnol. 2012; 52(2):129-139. Purification and analysis of integrity and components of VLPs Characterization of VLPs: nucleic acid content Next-generation sequencing (Ion Torrent system) RGMoV VLPs from P. pastoris Total reads: Identified sequences: 26 253 7 820 RGMoV CP mRNA: 4 678 pPIC3.5 ColE1 transcript: 1 102 P. pastoris Dienoyl-CoA isomerase (mitochondrial): 966 pPIC3.5 Km resistance: 563 P. pastoris chromosome 2 UTR: 346 Other (P. pastoris transcripts): 18 598 Purification and analysis of integrity and components of VLPs Bacteria Yeasts Insect Cells Mammalian Cells Plants Cell-free synthesis Typical workflow for purification and characterization of new VLPs - «Knowledge-based» buffer systems «Correct» cell disruption procedure Early identification: - analytical sucrose gradients - agarose gel analysis after nuclease treatment - electron microscopy Purification: - sucrose gradients; - column chromatography Characterization: - estimation of VLP concentrations; - mass spectrometry; - dynamic light scattering; - electron microscopy; - stability studies including storage conditions; - nucleic acid packaging studies; VLPs Purification and analysis of integrity and components of VLPs ________________ Dr. Ina Baļķe Gunta Reseviča Ieva Kalnciema Jeļena Šaripo Vilija Zeltiņa _____________________ Acknowledgements: ________________ Dr. Kaspars Tārs Dr. Andris Kazaks Dr. Ivars Petrovskis Dr. Velta Ose Dr. Dace Skrastiņa Dr. Dāvids Fridmanis Prof. Dr. Pauls Pumpēns _____________________ ____________________ Prof. Dr. Martin Bachmann (Zurich) _________________________
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