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22. Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

Author

Sasnauskas Kęstutis, Juozapaitis Mindaugas, Samuel Dhanraj, Čiplys Evaldas, and Slibinskas Rimantas

Subjects
Microbiology, QR1-502
Abstract

Abstract Background The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN) and measles hemagglutinin (MeH) in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Results Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A) and is closely associated with small heat shock proteins (sHsps) that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto) in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Conclusions Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of these recombinant proteins induces the UPR's cytosolic counterpart, the UPR-Cyto, which represent a subset of proteins involved in the heat-shock response. The involvement of eEF1A may explain the mechanism by which only large chaperones, but not small Hsps are upregulated during this stress response. Our study highlights important differences between viral surface protein expression in yeast and mammalian cells at the first stage of secretory pathway.

Published
2011
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28. Development, optimization and application of PCR-based methods for molecular characterization of transgenic inserts in genetically modified plants

Author

ŽVINGILA, DONATAS, PAŠAKINSKIENĖ, IZOLDA, JARMALAITĖ, SONATA, SRUOGA, ANIOLAS, LUBYS, ARVYDAS, SASNAUSKAS, KĘSTUTIS, PAULAUSKAS, ALGIMANTAS, LAZUTKA, JUOZAS RIMANTAS, MILCAMPS, ANNE, Vilnius University, Spalinskas, Rapolas, ŽVINGILA, DONATAS, PAŠAKINSKIENĖ, IZOLDA, JARMALAITĖ, SONATA, SRUOGA, ANIOLAS, LUBYS, ARVYDAS, SASNAUSKAS, KĘSTUTIS, PAULAUSKAS, ALGIMANTAS, LAZUTKA, JUOZAS RIMANTAS, MILCAMPS, ANNE, Vilnius University, and Spalinskas, Rapolas

Abstract

One of the fastest developing fields of biotechnology in the past three decades is that of genetically modified (GM) plants for industrial applications, as well as food and feed. The European Union (EU) has probably the most rigorous regulations for GMOs in the world and as such, the decisions made by the EU political institutions on regulatory and marketing actions heavily depend on scientific evidence. This doctoral dissertation deals with the molecular biology laboratory methods based on the most popular to date method polymerase chain reaction (PCR) used in the molecular characterization of the genetically modified organisms (GMOs). When studying the molecular structure of the GMO, two main interests of the nucleotide sequence are: (1) the actual sequence of the transgenic insert, and (2) the plant genomic DNA surrounding the synthetic DNA. Two distinct techniques of the Long Template PCR and Genome Walking were analyzed on the GMO DNA matrices. The LT-PCR was applied to obtain single uninterrupted fragment containing full transgenic insert of the selected GM events. The analysis of LT-PCR was continued with the application of a novel PCR additive called carbon nanotubes (CNTs) to investigate the impact of this nanomaterial on the specificity, efficiency and the product yield. The Genome Walking is a technique to obtain unknown DNA fragments adjacent to the known DNA. This doctoral dissertation seeks to explain in detail the development and optimization of a method Long... [to full text], Viena iš greičiausiai besivystančių biotechnologijos mokslo sričių pastaruosius tris dešimtmečius yra genetiškai modifikuotų (GM) augalų kūrimas ir jų naudojimas pramonėje, maistui ir pašarams. Europos Sąjunga turi bene griežčiausią GMO reglamentavimą visame pasaulyje, t.y. visi politiniai sprendimai dėl transgeninių augalų patekimo į rinką yra pagrįsti mokslinių tyrimų rezultatais. Šioje disertacijoje apžvelgiami populiariausiu molekulinės biologijos metodu, polimerazine grandinine reakcija (PGR), grįstos technologijos genetiškai modifikuotų organizmų (GMO) tyrimams. Tiriant molekulinę GMO struktūrą, didžiausias dėmesys yra kreipiamas į šių nukleotidinių sekų aptikimą: (1) transgeninio intarpo, bei (2) augalo genomo sekų aplink transgeninio intarpo įsiterpimo vietą. Naudojant GMO DNR matricas, buvo tirtos dvi skirtingos technologijos: ilgagrandė PGR (I-PGR) ir „pasivaikščiojimas genomu“ (PG). I-PGR metodu buvo siekiama nukopijuoti ir pagausinti vienalytį DNR fragmentą, kuriame būtų nukopijuotas visas transgeninis intarpas. Šio metodo taikymo tyrimas buvo tęsiamas ištiriant naujovišką PGR priedą, anglies nanovamzdelius (ANV). Šis reakcijos mišinio priedas, manoma, yra potencialus reakcijos specifiškumo ir efektyvumo stipriklis. PG yra technologija, įgalinanti nežinomų DNR sekų, prisijungusių prie žinomų sekų, aptikimą. Šioje daktaro disertacijoje smulkiai aprašomas PG metodo, kurį pavadinome LT-RADE, kūrimas ir optimalių reakcijų sąlygų parinkimas. Taip pat, sėkmingas metodo... [toliau žr. visą tekstą]

Published
2014

29. Genetiškai modifikuotų augalų transgeninių intarpų molekulinis apibūdinimas kuriant, optimizuojant ir taikant polimerazine grandinine reakcija pagrįstus metodus

Author

ŽVINGILA, DONATAS, PAŠAKINSKIENĖ, IZOLDA, JARMALAITĖ, SONATA, SRUOGA, ANIOLAS, LUBYS, ARVYDAS, SASNAUSKAS, KĘSTUTIS, PAULAUSKAS, ALGIMANTAS, LAZUTKA, JUOZAS RIMANTAS, MILCAMPS, ANNE, Vilnius University, Spalinskas, Rapolas, ŽVINGILA, DONATAS, PAŠAKINSKIENĖ, IZOLDA, JARMALAITĖ, SONATA, SRUOGA, ANIOLAS, LUBYS, ARVYDAS, SASNAUSKAS, KĘSTUTIS, PAULAUSKAS, ALGIMANTAS, LAZUTKA, JUOZAS RIMANTAS, MILCAMPS, ANNE, Vilnius University, and Spalinskas, Rapolas

Abstract

Viena iš greičiausiai besivystančių biotechnologijos mokslo sričių pastaruosius tris dešimtmečius yra genetiškai modifikuotų (GM) augalų kūrimas ir jų naudojimas pramonėje, maistui ir pašarams. Europos Sąjunga turi bene griežčiausią GMO reglamentavimą visame pasaulyje, t.y. visi politiniai sprendimai dėl transgeninių augalų patekimo į rinką yra pagrįsti mokslinių tyrimų rezultatais. Šioje disertacijoje apžvelgiami populiariausiu molekulinės biologijos metodu, polimerazine grandinine reakcija (PGR), grįstos technologijos genetiškai modifikuotų organizmų (GMO) tyrimams. Tiriant molekulinę GMO struktūrą, didžiausias dėmesys yra kreipiamas į šių nukleotidinių sekų aptikimą: (1) transgeninio intarpo, bei (2) augalo genomo sekų aplink transgeninio intarpo įsiterpimo vietą. Naudojant GMO DNR matricas, buvo tirtos dvi skirtingos technologijos: ilgagrandė PGR (I-PGR) ir „pasivaikščiojimas genomu“ (PG). I-PGR metodu buvo siekiama nukopijuoti ir pagausinti vienalytį DNR fragmentą, kuriame būtų nukopijuotas visas transgeninis intarpas. Šio metodo taikymo tyrimas buvo tęsiamas ištiriant naujovišką PGR priedą, anglies nanovamzdelius (ANV). Šis reakcijos mišinio priedas, manoma, yra potencialus reakcijos specifiškumo ir efektyvumo stipriklis. PG yra technologija, įgalinanti nežinomų DNR sekų, prisijungusių prie žinomų sekų, aptikimą. Šioje daktaro disertacijoje smulkiai aprašomas PG metodo, kurį pavadinome LT-RADE, kūrimas ir optimalių reakcijų sąlygų parinkimas. Taip pat, sėkmingas metodo... [toliau žr. visą tekstą], One of the fastest developing fields of biotechnology in the past three decades is that of genetically modified (GM) plants for industrial applications, as well as food and feed. The European Union (EU) has probably the most rigorous regulations for GMOs in the world and as such, the decisions made by the EU political institutions on regulatory and marketing actions heavily depend on scientific evidence. This doctoral dissertation deals with the molecular biology laboratory methods based on the most popular to date method polymerase chain reaction (PCR) used in the molecular characterization of the genetically modified organisms (GMOs). When studying the molecular structure of the GMO, two main interests of the nucleotide sequence are: (1) the actual sequence of the transgenic insert, and (2) the plant genomic DNA surrounding the synthetic DNA. Two distinct techniques of the Long Template PCR and Genome Walking were analyzed on the GMO DNA matrices. The LT-PCR was applied to obtain single uninterrupted fragment containing full transgenic insert of the selected GM events. The analysis of LT-PCR was continued with the application of a novel PCR additive called carbon nanotubes (CNTs) to investigate the impact of this nanomaterial on the specificity, efficiency and the product yield. The Genome Walking is a technique to obtain unknown DNA fragments adjacent to the known DNA. This doctoral dissertation seeks to explain in detail the development and optimization of a method Long... [to full text]

Published
2014

30. Prevalence of antibodies to hantavirus among hemodialysis patients with end-stage renal failure in Kaunas and its district

Author

Dargevičius, Arvydas, Petraitytė, Rasa, Sribikienė, Birutė, Šileikienė, Elvyra, Razukevičienė, Loreta, Žiginskienė, Edita, Vorobjovienė, Rita, Ražanskienė, Aušra, Sasnauskas, Kęstutis, Bumblytė, Inga Arūnė, and Kuzminskis, Vytautas

Subjects
Lėtinis inkstų nepakankamumas--Epidemiologija, Viruses, Virusai, Chronic renal failure--Epidemiology
Abstract

Tyrimo tikslas. Ištirti antikūnų prieš Hanta virusus paplitimą tarp Kauno ir Kauno apskrities ligonių, hemodializuojamų dėl galutinės stadijos lėtinio inkstų nepakankamumo. Tyrimo medžiaga ir metodai. Surinkti 218 pacientų kraujo serumai. Serumai paruošti Kauno medicinos universiteto klinikų Imunologijos laboratorijoje. Serumų analizė atlikta Biotechnolo¬gijos instituto Eukariotų genų inžinerijos laboratorijoje. Serumai pirmiausia buvo tikrinami netiesioginės imunofermentinės analizės būdu. Serumai, kurie imunofermentinės analizės metu teigiamai reagavo su Hanta virusų baltymais, buvo papildomai tikrinami imunoblotingo metodu. Testavimui buvo naudojami išgryninti rekombinantiniai mielėse ekspresuoti Hanta virusų nukleokapsidės baltymai. Rezultatai. Tarp 218 tirtų pacientų 16 (7,4 proc.) rasta antikūnų prieš Hanta virusus. Rasta antikūnų prieš Puumala ir Dobrava virusus: 13 (81 proc.) tiriamųjų nustatyta antikūnų prieš Dobrava virusą, 3 (18 proc.) rasta antikūnų prieš Puumala virusą. Tiriamųjų, kuriems rasta antikūnų prieš Hanta virusus, amžiaus vidurkis siekė 68±15,3 metų, o tiriamųjų, kuriems antikūnų prieš Hanta virusus nerasta, amžiaus vidurkis buvo 60,2±7,3 metų (p

Published
2007

31. Synthesis of hepatitis B virus surface protein derivates in yeast S. cerevisiae

Author

Bulavaitė, Aistė, Sabaliauskaitė, Rasa, Staniulis, Juozas Benediktas, and Sasnauskas, Kęstutis

Subjects
HBV surface proteins, udc:578, Hepatitis B virus, PreS1, Virus-like particles, B virusinis hepatitas, Virusologija, Baltymai
Abstract

HBV surface proteins PreS1[13–59]-S, PreS1[20–59]-S, PreS1[30–59]-S, PreS1[40–59]-S, PreS1[50–59]-S, PreS1[90–119]-S were produced in S.cerevisiae and purified. Electron microscopy suggested spherical virus-like particle formation for all the proteins except PreS1[90–119]-S. The PreS1[90–119] sequence was demonstrated to decrease protein solubility. Proteins are suitable for Tupaia primary hepatocyte binding investigations, diagnostic products and vaccine candidate development., Hepatito B viruo paviršiaus baltymai PreS1[13–59]-S, PreS1[20–59]-S, PreS1[30–59]-S, PreS1[40–59]-S, PreS1[50–59]-S, PreS1[90–119]-S susintetinti S. cerevisiae mielėse ir išgryninti. Baltymų elektroninė mikroskopija rodo, jog jie, išskyrus PreS1[90–119]-S, formuoja sferines, į virusą panašias daleles. PreS1[90–119] seka sumažina baltumų tirpumą. Baltymus galima panaudoti tiriant sąveiką su tupajų pirminiais hepatocitais, kuriant diagnostikos priemones, vakcinas.

Published
2006

32. Raudonukės viruso kapsidės baltymo sintezė mielėse ir jo charakterizavimas

Author

Petraitytė, Rasa and Sasnauskas, Kęstutis

Subjects
udc:577, Rubella virus, Recombinant capsid protein, Yeast, Indirect IgG ELISA
Abstract

In the present study, we expressed rubella virus (RV) capsid protein (C) in yeast S. cerevisiae cells. Two different methods for purification of recombinant C protein were employed: CsCl gradient ultracentrifugation and nickel chelation chromatography. The yield of recombinant C protein was approximately 3.2 mg of purified protein from 1g of wet yeast biomass. The antigenic characteristics of recombinant C protein purified in different ways were further evaluated by indirect IgG ELISA with RV-positive and RV-negative human serum specimens. Recombinant C protein purified using CsCl gradient ultracentrifugation possessed a higher antigenicity as compared to that purified by nickel chelate chromatography. The results indicate that the recombinant C protein has a potential for use in detection of human IgG antibodies against RV. The yeast-expressed rubella C protein is a promising antigen for the development of diagnostic tools in serology.

Published
2006

33. Generation of Recombinant Porcine Parvovirus Virus-Like Particles inSaccharomyces cerevisiaeand Development of Virus-Specific Monoclonal Antibodies

Author

Tamošiūnas, Paulius Lukas, primary, Petraitytė-Burneikienė, Rasa, additional, Lasickienė, Rita, additional, Akatov, Artiomas, additional, Kundrotas, Gabrielis, additional, Sereika, Vilimas, additional, Lelešius, Raimundas, additional, Žvirblienė, Aurelija, additional, and Sasnauskas, Kęstutis, additional

Published
2014
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34. Restrikcijos endonukleazių EcoRII, BfiI ir Bse634I struktūriniai ir funkciniai tyrimai

Author

Klimašauskas, Saulius, Sasnauskas, Kęstutis, Shakked, Zippora, Lubys, Arvydas, Lagunavičius, Arūnas, Venclovas, Česlovas, Merkienė, Eglė, Vilnius University, Golovenko, Dmitrij, Klimašauskas, Saulius, Sasnauskas, Kęstutis, Shakked, Zippora, Lubys, Arvydas, Lagunavičius, Arūnas, Venclovas, Česlovas, Merkienė, Eglė, Vilnius University, and Golovenko, Dmitrij

Abstract

Restrikcijos endonukleazės (REazės) – tai fermentai, kurie dėl savo ypatingo specifiškumo DNR taikiniui sulaukė plataus pritaikymo kaip DNR manipuliacijų in vitro ir genų inžinerijos įrankis. Kristalografiniais metodais nustatomos trimatės REazių atomų koordinačių struktūros (toliau – struktūros) padeda atskleisti, kaip skirtingos struktūrinės sanklodos yra pritaikytos restrikcijos endonukleazių funkcijai atlikti. Disertacijoje pristatomo darbo tikslas – kristalografiniais bei biocheminiais metodais ištirti specifiškumo-struktūros sąryšį PD-(D/E)XK ir fosfolipazių D (PLD) superšeimoms priklausantiems restrikcijos fermentams. Savo darbo objektais pasirinkome PD-(D/E)XK superšeimos Cfr10I/NgoMIV/Bse634I pogrupiui priklausančias REazes Bse634I ir EcoRII, bei PLD superšeimos fermentą BfiI. Disertacijoje pristatomos REazės Bse634I mutanto R226A komplekso su alternatyviais taikiniais 5'-ACCGGT ir 5'-GCCGGC struktūros atskleidė, kad Bse634I specifiškumą RCCGGY taikiniui lemia tiesioginio bei netiesioginio atpažinimo mechanizmai. REazės EcoRII N- ir C-galinių domenų kompleksų su specifine DNR struktūros iliustruoja du skirtingus taikinio 5'-CCWGG atpažinimo mechanizmus. EcoRII C-galinis domenas (EcoRII-C) išsuka centrinius A/T nukleotidus iš DNR duplekso. Ši deformacija leidžia fermentui panaudoti Cfr10I/NgoMIV/Bse634I REazių pogrupiui būdingus simetrinius konservatyvius struktūrinius elementus 5'-CCGG sekos atpažinimui. EcoRII N-galinio domeno (EcoRII-N) struktūra yra pirmas B3... [toliau žr. visą tekstą], Due to their unique specificity, restriction endonucleases (REases) have gained widespread application as indispensable tools for in vitro manipulation and cloning of DNA. Delineation of the repertoire of the protein folds, providing three-dimensional portraits of the REases by crystallographic methods, should reveal how different folds are tailored to function as restriction enzymes. The major goal of this work was to explore the specificity‑structure relationships within PD‑(D/E)XK and phospholipase D superfamily enzymes using a combination of the crystallographic and biochemical methods. More specifically, we have focused on the Bse634I and EcoRII restriction enzymes belonging to the Cfr10I/NgoMIV/Bse634I branch of PD‑(D/E)XK superfamily, and BfiI REase of PLD superfamily. Dissertation presents the crystal structure of the Bse634I REase mutant (R226A) complexed with two alternative target sites 5'‑ACCGGT and 5'‑GCCGGC, respectively. The analysis of the crystal structures revealed for the first time that the degenerate base pairs recognition by Bse634I is achieved through the combination of direct and indirect readout mechanisms. The crystal structures of the N‑ and C‑terminal domains of EcoRII solved in the DNA bound form revealed different structural mechanisms used for the recognition of the same target sequence 5'‑CCWGG. The first structural evidence has been provided that the C‑terminal domain of EcoRII (EcoRII-C) flips out the central nucleotides A/T while interacting... [to full text]

Published
2012

35. Structural and functional studies of restriction endonucleases EcoRII, BfiI and Bse634I

Author

Klimašauskas, Saulius, Sasnauskas, Kęstutis, Shakked, Zippora, Lubys, Arvydas, Lagunavičius, Arūnas, Venclovas, Česlovas, Merkienė, Eglė, Vilnius University, Golovenko, Dmitrij, Klimašauskas, Saulius, Sasnauskas, Kęstutis, Shakked, Zippora, Lubys, Arvydas, Lagunavičius, Arūnas, Venclovas, Česlovas, Merkienė, Eglė, Vilnius University, and Golovenko, Dmitrij

Abstract

Due to their unique specificity, restriction endonucleases (REases) have gained widespread application as indispensable tools for in vitro manipulation and cloning of DNA. Delineation of the repertoire of the protein folds, providing three-dimensional portraits of the REases by crystallographic methods, should reveal how different folds are tailored to function as restriction enzymes. The major goal of this work was to explore the specificity-structure relationships within PD-(D/E)XK and phospholipase D superfamily enzymes using a combination of the crystallographic and biochemical methods. More specifically, we have focused on the Bse634I and EcoRII restriction enzymes belonging to the Cfr10I/NgoMIV/Bse634I branch of PD-(D/E)XK superfamily, and BfiI REase of PLD superfamily. Dissertation presents the crystal structure of the Bse634I REase mutant (R226A) complexed with two alternative target sites 5'-ACCGGT and 5'-GCCGGC, respectively. The analysis of the crystal structures revealed for the first time that the degenerate base pairs recognition by Bse634I is achieved through the combination of direct and indirect readout mechanisms. The crystal structures of the N- and C-terminal domains of EcoRII solved in the DNA bound form revealed different structural mechanisms used for the recognition of the same target sequence 5'-CCWGG. The first structural evidence has been provided that the C-terminal domain of EcoRII (EcoRII-C) flips out the central nucleotides A/T while interacting... [to full text], Restrikcijos endonukleazės (REazės) – tai fermentai, kurie dėl savo ypatingo specifiškumo DNR taikiniui sulaukė plataus pritaikymo kaip DNR manipuliacijų in vitro ir genų inžinerijos įrankis. Kristalografiniais metodais nustatomos trimatės REazių atomų koordinačių struktūros (toliau – struktūros) padeda atskleisti, kaip skirtingos struktūrinės sanklodos yra pritaikytos restrikcijos endonukleazių funkcijai atlikti. Disertacijoje pristatomo darbo tikslas – kristalografiniais bei biocheminiais metodais ištirti specifiškumo-struktūros sąryšį PD-(D/E)XK ir fosfolipazių D (PLD) superšeimoms priklausantiems restrikcijos fermentams. Savo darbo objektais pasirinkome PD-(D/E)XK superšeimos Cfr10I/NgoMIV/Bse634I pogrupiui priklausančias REazes Bse634I ir EcoRII, bei PLD superšeimos fermentą BfiI. Disertacijoje pristatomos REazės Bse634I mutanto R226A komplekso su alternatyviais taikiniais 5'-ACCGGT ir 5'-GCCGGC struktūros atskleidė, kad Bse634I specifiškumą RCCGGY taikiniui lemia tiesioginio bei netiesioginio atpažinimo mechanizmai. REazės EcoRII N- ir C-galinių domenų kompleksų su specifine DNR struktūros iliustruoja du skirtingus taikinio 5'-CCWGG atpažinimo mechanizmus. EcoRII C-galinis domenas (EcoRII-C) išsuka centrinius A/T nukleotidus iš DNR duplekso. Ši deformacija leidžia fermentui panaudoti Cfr10I/NgoMIV/Bse634I REazių pogrupiui būdingus simetrinius konservatyvius struktūrinius elementus 5'-CCGG sekos atpažinimui. EcoRII N-galinio domeno (EcoRII-N) struktūra yra pirmas B3... [toliau žr. visą tekstą]

Published
2012

44. Generation of Recombinant Porcine Parvovirus Virus-Like Particles in Saccharomyces cerevisiae and Development of Virus-Specific Monoclonal Antibodies.

Author

Tamošiūnas, Paulius Lukas, Petraitytė-Burneikienė, Rasa, Lasickienė, Rita, Akatov, Artiomas, Kundrotas, Gabrielis, Sereika, Vilimas, Lelešius, Raimundas, Žvirblienė, Aurelija, and Sasnauskas, Kęstutis

Subjects
PARVOVIRUS diseases, NUCLEIC acid synthesis, CIRCOVIRUS diseases, VIREMIA, ELECTRON microscope techniques, THERAPEUTICS
Abstract

Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance. [ABSTRACT FROM AUTHOR]

Published
2014
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48. 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.

Author

Tamošiūnas, Paulius Lukas, Simutis, Karolis, Kodzė, Indrė, Firantienė, Regina, Ėmužytė, Regina, Petraitytė-Burneikienė, Rasa, Žvirblienė, aurelija, and Sasnauskas, Kęstutis

Subjects
PARVOVIRUSES, VIRUS-like particles, YEAST, VIRAL antibodies, VIRAL disease diagnosis, SACCHAROMYCES cerevisiae, IMMUNOSPECIFICITY
Abstract

Background: Human parvovirus 4 (PARV4) is a recently discovered member of the Parvoviridae family, which is not closely related to any previously discovered human parvoviruses. PARV4 has been isolated from the plasma of individuals with symptoms of acute viral infection; however, until recently PARV4 had not been associated with any disease, and its prevalence in the human population is yet to be established. Methods: The major capsid protein VP2 of PARV4 was generated in the yeast Saccharomyces cerevisiae and used for serological detection of virus-specific IgG and IgM in the sera of low-risk individuals. Results: One hundred and seventy serum specimens obtained from patients with acute respiratory diseases were tested for PARV4-specific IgG and IgM antibodies. Sixteen individuals (9.4%) were diagnosed as seropositive, including 6 IgM and IgG positive, 6 IgM positive/IgG negative and 4 IgG positive/IgM negative. Seven of the 16 seropositive individuals were between the ages of 3 and 11 with no evidence of parenteral exposure to PARV4 infection. Conclusion: Our data demonstrate that recombinant yeast-derived VP2 protein, self-assembled to virus-like particles, can represent a useful tool when studying the seroprevalence of PARV4 infection. The presence of PARV4-specific antibodies in a low-risk group may indicate the possibility of alternative routes of virus transmission. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Published
2013
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50. Expression and characterisation of rubella virus capsid protein in yeast cells.

Author

Petraitytė, Rasa and Sasnauskas, Kęstutis

Subjects
*RUBELLA virus, *TOGAVIRUSES, *PROTEINS, *YEAST, *ULTRACENTRIFUGATION
Abstract

In the present study, we expressed rubella virus (RV) capsid protein (C) in yeast S. cerevisiae cells. Two different methods for purification of recombinant C protein were employed: CsCl gradient ultracentrifugation and nickel chelation chromatography. The yield of recombinant C protein was approximately 3.2 mg of purified protein from 1g of wet yeast biomass. The antigenic characteristics of recombinant C protein purified in different ways were further evaluated by indirect IgG ELISA with RV-positive and RV-negative human serum specimens. Recombinant C protein purified using CsCl gradient ultracentrifugation possessed a higher antigenicity as compared to that purified by nickel chelate chromatography. The results indicate that the recombinant C protein has a potential for use in detection of human IgG antibodies against RV. The yeast-expressed rubella C protein is a promising antigen for the development of diagnostic tools in serology. [ABSTRACT FROM AUTHOR]

Published
2006

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