Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles |
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Authors: | Leona Gilbert Jouni Toivola Outi Välilehto Taija Saloniemi Claire Cunningham Daniel White Anna R Mäkelä Eila Korhonen Matti Vuento Christian Oker-Blom |
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Institution: | 1. Department of Biological and Environmental Science and Nanoscience Center, University of Jyv?skyl?, P.O. Box 35, 40014, Finland
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Abstract: | Fluorescence correlation spectroscopy (FCS) monitors random movements of fluorescent molecules in solution, giving information
about the number and the size of for example nano-particles. The canine parvovirus VP2 structural protein as well as N-terminal
deletion mutants of VP2 (-14, -23, and -40 amino acids) were fused to the C-terminus of the enhanced green fluorescent protein
(EGFP). The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy
as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants
truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that
the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were
able to form virus-like particles (VLPs). The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat
larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated
by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by
confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was
determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information
for strategic development of parvovirus-like particles. |
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