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Characterization and purification of pentameric chimeric protein particles using asymmetric flow field-flow fractionation coupled with multiple detectors
Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence of commercial vaccines, the development of more effective and cheaper vaccines is expected. The usage of chimeric antigens allows serological differentiation between naturally infected and vaccinated an...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer Berlin Heidelberg
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035888/ https://www.ncbi.nlm.nih.gov/pubmed/33837800 http://dx.doi.org/10.1007/s00216-021-03323-6 |
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author | Kotoucek, Jan Hezova, Renata Vrablikova, Alena Hubatka, Frantisek Kulich, Pavel Macaulay, Stuart Roessner, Dierk Raska, Milan Psikal, Ivan Turanek, Jaroslav |
author_facet | Kotoucek, Jan Hezova, Renata Vrablikova, Alena Hubatka, Frantisek Kulich, Pavel Macaulay, Stuart Roessner, Dierk Raska, Milan Psikal, Ivan Turanek, Jaroslav |
author_sort | Kotoucek, Jan |
collection | PubMed |
description | Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence of commercial vaccines, the development of more effective and cheaper vaccines is expected. The usage of chimeric antigens allows serological differentiation between naturally infected and vaccinated animals. In this work, recombinant pentameric vaccination protein particles spontaneously assembled from identical subunits-chimeric fusion proteins derived from circovirus capsid antigen Cap and a multimerizing subunit of mouse polyomavirus capsid protein VP1 were purified and characterized using asymmetric flow field-flow fractionation (AF4) coupled with UV and MALS/DLS (multi-angle light scattering/dynamic light scattering) detectors. Various elution profiles were tested, including constant cross-flow and decreasing cross-flow (linearly and exponentially). The optimal sample retention, separation efficiency, and resolution were assessed by the comparison of the hydrodynamic radius (R(h)) measured by online DLS with the R(h) values calculated from the simplified retention equation according to the AF4 theory. The results show that the use of the combined elution profiles (exponential and constant cross-flow rates) reduces the time of the separation, prevents undesirable sample-membrane interaction, and yields better resolution. Besides, the results show no self-associations of the individual pentameric particles into larger clusters and no sample degradation during the AF4 separation. The R(g)/R(h) ratios for different fractions are in good correlation with morphological analyses performed by transmission electron microscopy (TEM). Additionally to the online analysis, the individual fractions were subjected to offline analysis, including batch DLS, TEM, and SDS-PAGE, followed by Western blot. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00216-021-03323-6. |
format | Online Article Text |
id | pubmed-8035888 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Springer Berlin Heidelberg |
record_format | MEDLINE/PubMed |
spelling | pubmed-80358882021-04-12 Characterization and purification of pentameric chimeric protein particles using asymmetric flow field-flow fractionation coupled with multiple detectors Kotoucek, Jan Hezova, Renata Vrablikova, Alena Hubatka, Frantisek Kulich, Pavel Macaulay, Stuart Roessner, Dierk Raska, Milan Psikal, Ivan Turanek, Jaroslav Anal Bioanal Chem Research Paper Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence of commercial vaccines, the development of more effective and cheaper vaccines is expected. The usage of chimeric antigens allows serological differentiation between naturally infected and vaccinated animals. In this work, recombinant pentameric vaccination protein particles spontaneously assembled from identical subunits-chimeric fusion proteins derived from circovirus capsid antigen Cap and a multimerizing subunit of mouse polyomavirus capsid protein VP1 were purified and characterized using asymmetric flow field-flow fractionation (AF4) coupled with UV and MALS/DLS (multi-angle light scattering/dynamic light scattering) detectors. Various elution profiles were tested, including constant cross-flow and decreasing cross-flow (linearly and exponentially). The optimal sample retention, separation efficiency, and resolution were assessed by the comparison of the hydrodynamic radius (R(h)) measured by online DLS with the R(h) values calculated from the simplified retention equation according to the AF4 theory. The results show that the use of the combined elution profiles (exponential and constant cross-flow rates) reduces the time of the separation, prevents undesirable sample-membrane interaction, and yields better resolution. Besides, the results show no self-associations of the individual pentameric particles into larger clusters and no sample degradation during the AF4 separation. The R(g)/R(h) ratios for different fractions are in good correlation with morphological analyses performed by transmission electron microscopy (TEM). Additionally to the online analysis, the individual fractions were subjected to offline analysis, including batch DLS, TEM, and SDS-PAGE, followed by Western blot. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00216-021-03323-6. Springer Berlin Heidelberg 2021-04-10 2021 /pmc/articles/PMC8035888/ /pubmed/33837800 http://dx.doi.org/10.1007/s00216-021-03323-6 Text en © Springer-Verlag GmbH Germany, part of Springer Nature 2021 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. |
spellingShingle | Research Paper Kotoucek, Jan Hezova, Renata Vrablikova, Alena Hubatka, Frantisek Kulich, Pavel Macaulay, Stuart Roessner, Dierk Raska, Milan Psikal, Ivan Turanek, Jaroslav Characterization and purification of pentameric chimeric protein particles using asymmetric flow field-flow fractionation coupled with multiple detectors |
title | Characterization and purification of pentameric chimeric protein particles using asymmetric flow field-flow fractionation coupled with multiple detectors |
title_full | Characterization and purification of pentameric chimeric protein particles using asymmetric flow field-flow fractionation coupled with multiple detectors |
title_fullStr | Characterization and purification of pentameric chimeric protein particles using asymmetric flow field-flow fractionation coupled with multiple detectors |
title_full_unstemmed | Characterization and purification of pentameric chimeric protein particles using asymmetric flow field-flow fractionation coupled with multiple detectors |
title_short | Characterization and purification of pentameric chimeric protein particles using asymmetric flow field-flow fractionation coupled with multiple detectors |
title_sort | characterization and purification of pentameric chimeric protein particles using asymmetric flow field-flow fractionation coupled with multiple detectors |
topic | Research Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035888/ https://www.ncbi.nlm.nih.gov/pubmed/33837800 http://dx.doi.org/10.1007/s00216-021-03323-6 |
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