Department of Eukaryote Gene Engineering

EGE

Head: Dr. Rasa Burneikienė-Petraitytė

Tel. +370-5-2234421
Fax. +370-5-2687009
E-mail: rasa.burneikiene @ bti.vu.lt

 

Habil. Dr. Alma Gedvilaitė’s group activities:

The activities of the research group addresses studies related to efficient production of recombinant viral proteins in yeast cells by improvement of expression systems and yeast genetic background. Despite that yeast Saccharomyces cerevisiae frequently serves as a reasonable host for heterologous protein expression, in many instances, it is far from optimal. Our aim is to understand and balance processes in yeast trigged by synthesis of recombinant proteins, identify factors necessary for efficient recombinant protein expression by overexpression or deletion of target genes. In an attempt to elucidate the requirement of factors for the biosynthesis of recombinant viral and human proteins we use proteomics, yeast mutant and gene collection studies. Very important part of group study is related to improvement of yeast protein secretion capacities by identification and investigation of yeast genes conferring supersecretion phenotypes.

The group is also interested in search and characterization of new polyomaviruses, studies of virus-like particles (VLPs) formation, and protein engineering based on construction of chimeric VLPs harboring foreign epitopes. We employ hamster polyomavirus VLPs as carriers for short and non-immunogenic protein sequences with are presented on VP1 VLPs to increase their immunogenicity. For presentation of large and complex protein molecules (as single chain antibody molecules) we use VP1/VP2 VLPs.

We have a wide network of international collaboration and long-lasting experience in conducting collaborative projects with industrial partners.

Current project:

Project of Research Council of Lithuania "Investigation of Kluyveromyces lactis mutations conferring enhanced secretion phenotype and generation of yeast strains for supersecretion of recombinant proteins", 2017-2021, project leader A.Gedvilaite.

Results:

  • Identified four new common and bank voles, yellow-necked mouse and shrews polyomaviruses.

  • Large collection of more than 40 different virus like particles (VLPs) derived from various polyomavirus (all 13 human polyomavirus, rodents, avian and other host polyomavirus) VP1 proteins and papillomavirus L1 proteins (HPV6, 11, 16, 18, 31, 33), porcine circovirus type 2 capsid proteins.

  • Yeast expression systems for construction and production of hamster polyomavirus VP1 and VP1/VP2 VLPs as carriers harboring non immunogenic peptides or complex molecules of interest.

  • Genetic engineered yeast strain collection for recombinant protein production.

  • 2003 – Lithuanian Science Prize in the Biomedicine Sciences area.

  • Patent: Zvirblienė A., Gedvilaitė A., Ulrich, R., Sasnauskas. Process for the production of monoclonal antibodies using chimeric VLPs. US Patent No.:US7,919,314 B2. Apr. 5, 2011.

Selected publications:

  1. Gedvilaite A, Tryland M, Ulrich RG, Schneider J, Kurmauskaite V, Moens U, Preugschas H, Calvignac-Spencer S, Ehlers B. Novel polyomaviruses in shrews (Soricidae) with close similarity to human polyomavirus 12. J Gen Virol. 2017. doi: 10.1099/jgv.0.000948.

  2. Zaveckas M, Goda K, Ziogiene D, Gedvilaite A. Purification of recombinant trichodysplasia spinulosa-associated polyomavirus VP1-derived virus-like particles using chromatographic techniques. J Chromatogr B Analyt Technol Biomed Life Sci. 2018 May 9;1090:7-13. doi: 10.1016/j.jchromb.2018.05.007.

  3. Valaviciute M, Norkiene M, Goda K, Slibinskas R, Gedvilaite A. Survey of molecular chaperone requirement for the biosynthesis of hamster polyomavirus VP1 protein in Saccharomyces cerevisiae. Arch Virol. 2016. 161(7), 1807-1819. DOI 10.1007/s00705-016-2846-3

  4. Nainys J, Timinskas A, Schneider J, Ulrich RG, Gedvilaite A. Identification of Two Novel Members of the Tentative Genus Wukipolyomavirus in Wild Rodents. PLoS One. 2015 Oct 16;10(10):e0140916. doi: 10.1371/journal.pone.0140916. eCollection 2015.

  5. Pleckaityte M, Bremer CM, Gedvilaite A, Kucinskaite-Kodze I, Glebe D, Zvirbliene A. Construction of polyomavirus-derived pseudotype virus-like particles displaying a functionally active neutralizing antibody against hepatitis B virus surface antigen. BMC Biotechnology. 2015, 15:85. DOI 10.1186/s12896-015-0203-3

  6. Norkiene M, Stonyte J, Ziogiene D, Mazeike E, Sasnauskas K, Gedvilaite A. Production of recombinant VP1-derived virus-like particles from novel human polyomaviruses in yeast. BMC Biotechnology. 2015, 15:68. DOI: 10.1186/s12896-015-0187-z

  7. Zaveckas M., Snipaitis S., Pesliakas H. Nainys J. Gedvilaite A. Purification of recombinant virus-like particles of porcine circovirustype 2 capsid protein using ion-exchange monolith chromatography. J Chromatogr B. 2015, v. 991, p.21–28. doi:10.1016/j.jchromb.2015.04.004

  8. Nainys J, Lasickiene R, Petraityte-Burneikiene R, Dabrisius J, Lelesius R, Sereika V, Zvirbliene A, Sasnauskas K, Gedvilaite A. 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 Biotechnology 2014 14:100. doi:10.1186/s12896-014-0100-1 847

  9. Pleckaityte M, Zvirbliene A, Sezaite I, Gedvilaite A. Production in yeast of pseudotype virus-like particles harboring functionally active antibody fragments neutralizing the cytolytic activity of vaginolysin. Microb Cell Fact. 2011. 10:109.

  10. Gedvilaite A., Dorn D.C., Sasnauskas K., Pecher G., Bulavaite A., Lawatscheck R., Staniulis J., Dalianis T., Ramqvist T., Schönrich G., Raftery MJ., Ulrich R. Virus-like particles derived from major capsid protein VP1 of different polyomaviruses differ in their ability to induce maturation in human dendritic cells. Virology, 2006, v. 354 (2), 252-260.

  11. Gedvilaite A., Frommel C., Sasnauskas K., Burkhard M., Ozel M., Behrsing O., Staniulis J., Jandrig B., Scherneck S., Ulrich R. Formation of Immunogenic Virus-like Particles by Inserting Epitopes into Surface-Exposed regions of Hamster Major Capsid Protein // Virology. 2000, v. 273, n. 1, p. 21-35.

 

Group of Drs. Rimantas Slibinskas and Evaldas Čiplys

Research:

Our group is studying processes of recombinant protein synthesis in yeast expression systems by using various methods including proteomic analysis. Research is mainly focused on foreign protein processing in cell secretion pathway and related stress responses. Essential molecular components for proper functioning of secretory pathway and for efficient secretion of recombinant proteins are chaperones located in endoplasmic reticulum (ER). We found that human ER chaperones are correctly processed and efficiently produced in the active form in yeast cells suggesting compatibility of human and yeast cellular machinery for synthesis and processing of these specific proteins. Moreover, yeast cells secreted large amounts of human ER chaperones BiP/GRP78, calreticulin and ERp57 under conditions of overexpression. It is known that translocation of these proteins to the cell surface or their secretion outside the cells in human tissues is related to various pathological conditions with connection to a number of human diseases. Therefore, it makes yeast expression systems an excellent model for studying molecular mechanisms of the secretion of human ER chaperones. Furthermore, high quality and widely affordable recombinant human ER chaperone proteins are needed for further research and studies of the potential application of these proteins. For this purpose we also develop technologies for the production of human ER chaperones and some of their mutants in yeast.

 

Current project:

Research and development project, partially supported by the EU structural funds, „Development of technology for production of native recombinant human calreticulin in yeast“ (2016-2019), project leaders R. Slibinskas and E. Čiplys.

 

Selected publications:

  1. Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae. Ciplys E, Samuel D, Juozapaitis M, Sasnauskas K, Slibinskas R.Microb Cell Fact. 2011 May 19;10:37. doi: 10.1186/1475-2859-10-37.

  1. Overexpression of human calnexin in yeast improves measles surface glycoprotein solubility. Ciplys E, Sasnauskas K, Slibinskas R.FEMS Yeast Res. 2011 Sep;11(6):514-23. doi: 10.1111/j.1567-1364.2011.00742.x. Epub 2011 Jun 27.

  1. Native signal peptide of human ERp57 disulfide isomerase mediates secretion of active native recombinant ERp57 protein in yeast Saccharomyces cerevisiae.Čiplys E, Žitkus E, Slibinskas R. Protein Expr Purif. 2013 Jun;89(2):131-5. doi: 10.1016/j.pep.2013.03.003. Epub 2013 Mar 23.

  1. Comparison of first dimension IPG and NEPHGE techniques in two-dimensional gel electrophoresis experiment with cytosolic unfolded protein response in Saccharomyces cerevisiae. Slibinskas R, Ražanskas R, Zinkevičiūtė R, Čiplys E. Proteome Sci. 2013 Jul 27;11(1):36. doi: 10.1186/1477-5956-11-36.

  1. Generation of human ER chaperone BiP in yeast Saccharomyces cerevisiae. Čiplys E, Aučynaitė A, Slibinskas R. Microb Cell Fact. 2014 Feb 11;13:22.doi: 10.1186/1475-2859-13-22.

  1. Calreticulin as cancer treatment adjuvant: combination with photodynamic therapy and photodynamic therapy-generated vaccines. Korbelik M, Banáth J, Saw KM, Zhang W, Čiplys E. Front Oncol. 2015 Feb 3;5:15. doi: 10.3389/fonc.2015.00015.

  1. Heat shock at higher cell densities improves measles hemagglutinin translocation and human GRP78/BiP secretion in Saccharomyces cerevisiae. Zinkevičiūtė R, Bakūnaitė E, Čiplys E, Ražanskas R, Raškevičiūtė J, Slibinskas R. N Biotechnol. 2015 Dec 25;32(6):690-700. doi: 10.1016/j.nbt.2015.04.001. Epub 2015 Apr 20.

  2. High-level secretion of native recombinant human calreticulin in yeast. Čiplys E, Žitkus E, Gold LI, Daubriac J, Pavlides SC, Højrup P, Houen G, Wang WA, Michalak M, Slibinskas R. Microb Cell Fact. 2015 Oct 15;14:165.doi: 10.1186/s12934-015-0356-8.

  1. Mapping the Ca(2+) induced structural change in calreticulin. Boelt SG, Norn C, Rasmussen MI, André I, Čiplys E, Slibinskas R, Houen G, Højrup P. J Proteomics. 2016 Jun 16;142:138-48. doi: 10.1016/j.jprot.2016.05.015. Epub 2016 May 16.

  1. Survey of molecular chaperone requirement for the biosynthesis of hamster polyomavirus VP1 protein in Saccharomyces cerevisiae. Valaviciute M, Norkiene M, Goda K, Slibinskas R, Gedvilaite A. Arch Virol. 2016 Jul;161(7):1807-19. doi: 10.1007/s00705-016-2846-3.

 

Group of Dr. Rasa Petraitytė-Burneikienė

Analysis, synthesis of viral proteins in yeast and their practical application is the main research field of our group. We are focusing on aspects related to the production of recombinant proteins in yeast expression systems, the development and optimization of expression systems dedicated to the production of recombinant proteins as virus-like particles. The core research techniques used in the department are the methods of molecular biology and biotechnology, leading to obtain recombinant plasmids, genetically modified bacteria and yeast, and purification of proteins. We have already in our disposition powerful diagnostic tools as yeast-expressed recombinant proteins for diagnostics of human hantaviruses, porcine parvovirus, human bocaviruses 1-4, human metapneumovirus, human parvovirus 4, parainfluenza viruses 2 and 4, hepatitis E and bovine Schmallenberg virus.  Yeast expressed recombinant proteins are applied in the tests for detection of virus-specific antibodies in human serum and oral fluid samples.

 

Current projects:

1.    National programme “Healthy Aging”, Research Council of Lithuania. Investigation of synthesis regulation of proteins associated with Alzheimer disease development (No. SEN-05/2015). K. Sasnauskas. 2015–2018.

2.   Joint Lithuanian–Latvian–Chinese (Taiwanese) Tripartite Cooperation Programme. Research Council of Lithuania. Studying of human parvovirus B19, bocavirus and parvovirus 4 involvement in inflammatory neurological diseases using interdisciplinary approach (No. TAP LLT-3/2017). R. Petraitytė-Burneikienė. 2017-2019.

4.    LSC project for activity according action 2015-LMT-K-718 “Scientific research for advanced investigators“ (Smart specialization) , No. 01.2.2-LMT-K-718-01-0008 ,,New technologies for construction of recombinant allergens“, G. Žvirblis, 2018-2021.

 

Collaboration

Friedrich-Loeffler-Institut Bundesforschungsinstitut für Tiergesundheit, Federal Research

Institute for Animal Health OIE Collaborating Centre for Zoonoses in Europe (Germany)

Department of Virology, University of Freiburg (Germany)

A. Kirchenstein Institute of Microbiology and Virology, Riga Stradins University (Latvia)

Department of Medical Research, Mackay Memorial Hospital, (Taiwan)

 

Selected publications:

1.        Bulavaitė A, Lasickienė R, Vaitiekaitė A, Sasnauskas K, Žvirblienė A. (2016). Synthesis of human parainfluenza virus 2 nucleocapsid protein in yeast as nucleocapsid-like particles and investigation of its antigenic structure. Appl Microbiol Biotechnol. 100(10):4523-34.

2.        Chandy, S., Ulrich, R.G., Schlegel, M., Petraityte, R., Sasnauskas, K., Prakash, D.J., Balraj, V., Abraham, P., Sridharan, G. (2013). Hantavirus Infection among Wild Small Mammals in Vellore, South India. Zoonoses Public Health. 60(5):336-40.

3.        Dargevicius, A., Petraityte, R., Sribikiene, B., Sileikiene, E., Razukeviciene, L., Ziginskiene, E., Vorobjoviene, R., Razanskiene, A., Sasnauskas, K., Bumblyte, I.A., Kuzminskis, V.I. (2007). Prevalence of antibodies to hantavirus among hemodialysis patients with end-stage renal failure in Kaunas and its district. Medicina (Kaunas). 43 Suppl. 1:72-6.

4.        Emuzyte, R., Firantiene, R., Petraityte, R., Sasnauskas, K.  (2009). Human rhinoviruses, allergy, and asthma: a clinical approach. Medicina (Kaunas).  45(11):839-47.

5.        Essbauer, S.S., Schmidt-Chanasit, J., Madeja, E.L., Wegener, W., Friedrich, R., Petraityte, R., Sasnauskas, K., Jacob, J., Koch, J., Dobler, G., Conraths, F.J., Pfeffer, M., Pitra, C., and Ulrich, R.G. (2007). Nephropathia epidemica in metropolitan area, Germany. Emerg. Infect. Dis. 13(8):1271-1273.

6.        Gedvilaitė A., Dorn D.C., Sasnauskas K., Pecher G., Bulavaitė A., Lawatscheck R., Staniulis J., Dalianis T., Ramqvist T., Schönrich G., Raftery M.J. and Ulrich,R.  Virus-like particles derived from major capsid protein VP1 of different polyomaviruses differ in their entry pathways and ability to induce maturation in human dendritic cells. Virology // ISSN: 0042-6822, Academic Press, New York, 2006,354 (2), 252-260.

7.        Kucinskaite-Kodze I, Pleckaityte M, Bremer CM, Seiz PL, Zilnyte M, Bulavaite A, Mickiene G, Zvirblis G, Sasnauskas K, Glebe D, Zvirbliene A. (2016). New broadly reactive neutralizing antibodies against hepatitis B virus surface antigen. Virus Res. 211:209-21.

8.        Kucinskaite-Kodze, I., Petraityte-Burneikiene, R., Zvirbliene, A., Hjelle, B., Medina, R.A., Gedvilaite, A., Razanskiene, A., Schmidt-Chanasit, J., Mertens, M., Padula, P., Sasnauskas, K., Ulrich, R.G. (2011). Characterization of monoclonal antibodies against hantavirus nucleocapsid protein and their use for immunohistochemistry on rodent and human samples. Arch Virol. 156(3):443-56

9.        Lazutka, J., Spakova, A., Sereika, V., Lelesius, R., Sasnauskas, K., Petraityte-Burneikiene, R. (2015). Saliva as an alternative specimen for detection of Schmallenberg virus-specific antibodies in bovines. BMC Veterinary Research. 11(1):237

10.     Lazutka, J., Zvirbliene, A., Dalgediene, I., Petraityte-Burneikiene, R., Spakova, A., Sereika, V., Lelesius, R., Wernike, K., Beer, M., Sasnauskas, K. (2014). Generation of Recombinant Schmallenberg Virus Nucleocapsid Protein in Yeast and Development of Virus-Specific Monoclonal Antibodies. Journal of Immunology Research. 2014:160316.

11.     Mertens, M., Hofmann, J., Petraityte-Burneikiene, R., Ziller, M., Sasnauskas, K., Friedrich, R., Niederstrasser, O., Krüger, D.H., Groschup, M.H., Petri, E., Werdermann, S., Ulrich, R.G. (2011). Seroprevalence study in forestry workers of a non-endemic region in eastern Germany reveals infections by Tula and Dobrava-Belgrade hantaviruses. Med. Microbiol. Immunol. 200(4):263-8.

12.     Mertens, M., Kindler, E., Emmerich, P., Esser, J., Wagner-Wiening, C., Wölfel, R., Petraityte-Burneikiene, R., Schmidt-Chanasit, J., Zvirbliene, A., Groschup, M.H., Dobler, G., Pfeffer, M., Heckel, G., Ulrich, R.G., Essbauer, S.S. (2011). Phylogenetic analysis of Puumala virus subtype Bavaria, characterization and diagnostic use of its recombinant nucleocapsid protein. Virus Genes. 43(2):177-91.

13.     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 Biotechnology. 9;14(1):100.

14.     Petraitytė, R., Jin, L., Hunjan, R., Ražanskienė, A., Žvirblienė, A., Sasnauskas, K. (2007). Use of Saccharomyces cerevisiae-expressed recombinant nucleocapsid protein to detect Hantaan virus-specific immunoglobulinG (IgG) and IgM in oral fluid. Clin. Vaccine Immunol. 14(12):1603-1608.

15.     Petraityte, R., Tamosiunas, P.L., Juozapaitis, M., Zvirbliene, A., Sasnauskas, K., Shiell, B., Russell, G., Bingham, J., Michalski, W.P. (2009). Generation of Tioman virus nucleocapsid-like particles in yeast Saccharomyces cerevisiae. Virus Res. 145(1):92-6.

16.     Petraityte, R., Yang, H., Hunjan, R., Razanskiene, A., Dhanilall, P., Ulrich, R.G., Sasnauskas, K., Jin, L. (2008).  Development and evaluation of serological assays for detection of Hantaanvirus-specific antibodies in human sera using yeast-expressed nucleocapsid protein. J. Virol. Methods. 148(1-2):89-95.

17.     Petraitytė-Burneikienė, R., Nalivaiko, K., Lasickienė, R., Firantienė, R., Emužytė, R., Sasnauskas, K., Zvirblienė, A. (2011). Generation of recombinant metapneumovirus nucleocapsid protein as nucleocapsid-like particles and development of virus-specific monoclonal antibodies. Virus Res. 161(2):131-9.

18.     Schlegel, M., Tegshduuren, E., Yoshimatsu, K., Petraityte, R., Sasnauskas, K., Hammerschmidt, B., Friedrich, R., Mertens, M., Groschup, M.H., Arai, S., Endo, R., Shimizu, K., Koma, T., Yasuda, S., Ishihara, C., Ulrich, R.G., Arikawa, J., Köllner, B. (2012). Novel serological tools for detection of Thottapalayam virus, a Soricomorpha-borne hantavirus. Arch. Virol. 157(11):2179-87.

19.     Schmidt, J., Meisel, H., Capria, S.G., Petraityte, R., Lundkvist, A., Hjelle, B., Vial, P.A., Padula, P., Kruger, D.H., Ulrich, R. (2006). Serological assays for the detection of human andes hantavirus infections based on its yeast-expressed nucleocapsid protein. Intervirology. 49(3):173-84.

20.     Schmidt-Chanasit, J., Essbauer, S., Petraityte, R., Yoshimatsu, K., Tackmann, K., Conraths, F.J., Sasnauskas, K., Arikawa, J., Thomas, A., Pfeffer, M., Scharninghausen, J.J., Splettstoesser, W., Wenk, M., Heckel, G., Ulrich R.G. (2010). Extensive host sharing of central European Tula virus. J Virol. 84(1):459-74.

21.     Skrastina, D., Bulavaite, A. Sominskaja, I., Kovalevska, L., Ose, V., Priede, D., Pumpens, P., Sasnauskas, K. Immunological behaviour of immunodominant hepatitis B virus envelope exposed on the surface of two different virus-like particle carriers. Vaccine, 2008, 26, 1972-1981.

22.     Skrastina, D., Petrovskis, I., Petraityte, R., Sominskaya, I., Ose, V., Lieknina, I., Bogans, J., Sasnauskas, K., Pumpens, P. (2013). Chimeric derivatives of hepatitis B virus core particles carrying major epitopes of the rubella virus E1 glycoprotein. Clin. Vaccine Immunol. 20(11):1719-28.

23.     Tamošiūnas, P. L., Petraitytė-Burneikienė, R., Lasickienė, R., Akatov, A., Kundrotas, G., Sereika, V., Lelešius, R., Zvirblienė, A., Sasnauskas, K. (2014). Generation of Recombinant Porcine Parvovirus Virus-Like Particles in Saccharomyces cerevisiae and Development of Virus-Specific Monoclonal Antibodies. Journal of Immunology Research. 2014:573531.

24.     Tamošiūnas, P. L., Simutis, K., Kodzė, I., Firantienė, R., Emužytė, R., Petraitytė-Burneikienė, R., Zvirblienė, A., Sasnauskas, K. (2013). Production of Human Parvovirus 4 VP2 Virus-Like Particles in Yeast and Their Evaluation as an Antigen for Detection of Virus-Specific Antibodies in Human Serum. Intervirology. 56(5):271-7.

25.     Tamošiūnas, P.L., Petraitytė-Burneikienė, R., Bulavaitė, A., Marcinkevičiūtė, K., Simutis, K., Lasickienė, R., Firantienė, R., Ėmužytė, R., Žvirblienė, A., Sasnauskas, K. (2016). Yeast-generated virus-like particles as antigens for detection of human bocavirus 1-4 specific antibodies in human serum. Applied Microbiology and Biotechnology. 100(11):4935-46.

26.     Zvirbliene, A., Kucinskaite-Kodze, I., Razanskiene, A., Petraityte-Burneikiene, R., Klempa, B., Ulrich, R.G., Gedvilaite, A. (2014). The Use of Chimeric Virus-like Particles Harbouring a Segment of Hantavirus Gc Glycoprotein to Generate a Broadly-Reactive Hantavirus-Specific Monoclonal Antibody. Viruses. 6, 640-660.

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