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Characterization and purification of pentameric chimeric protein particles using asymmetric flow field-flow fractionation coupled with multiple detectors
- Source :
- Analytical and Bioanalytical Chemistry
- Publication Year :
- 2021
- Publisher :
- Springer Berlin Heidelberg, 2021.
-
Abstract
- 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 (Rh) measured by online DLS with the Rh 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 Rg/Rh 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.
- Subjects :
- Circovirus
Recombinant Fusion Proteins
Recombinant antigen
02 engineering and technology
Fractionation
01 natural sciences
Biochemistry
Light scattering
Analytical Chemistry
Cell Line
Mice
Viral Proteins
Dynamic light scattering
Theilovirus
Animals
biology
Chemistry
Elution
010401 analytical chemistry
021001 nanoscience & nanotechnology
biology.organism_classification
Fusion protein
Fractionation, Field Flow
0104 chemical sciences
Asymmetric flow field flow fractionation
Porcine circovirus
Biophysics
Asymmetric flow field-flow fractionation (AF4)
Protein Multimerization
0210 nano-technology
Research Paper
Subjects
Details
- Language :
- English
- ISSN :
- 16182650 and 16182642
- Database :
- OpenAIRE
- Journal :
- Analytical and Bioanalytical Chemistry
- Accession number :
- edsair.doi.dedup.....72fc0ee0cd57fa29a7dcaada1dfdcf25