In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO) fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few microns long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here - providing a large amount of the viral capsid protein in the native form with relatively simple, rapid, and economical procedures - opens a new route toward large-scale production of a more efficient antigenic compound to be used as a vaccination tool or as an adjuvant, and also represents a top-quality biomaterial to be further modified for biotechnological purposes.

Bugli, F., Caprettini, V., Cacaci, M., Martini, C., Paroni Sterbini, F., Torelli, R., Della Longa, S., Papi, M., Palmieri, V., Giardina, B., Posteraro, B., Sanguinetti, M., Arcovito, A., Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein, <<INTERNATIONAL JOURNAL OF NANOMEDICINE>>, 2014; 9 (Maggio): 2727-2739. [doi:10.2147/IJN.S60014] [http://hdl.handle.net/10807/58553]

Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein

Bugli, Francesca;Cacaci, Margherita;Martini, Cecilia;Paroni Sterbini, Francesco;Torelli, Riccardo;Papi, Massimiliano;Palmieri, Valentina;Giardina, Bruno;Posteraro, Brunella;Sanguinetti, Maurizio;Arcovito, Alessandro
2014

Abstract

In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO) fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few microns long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here - providing a large amount of the viral capsid protein in the native form with relatively simple, rapid, and economical procedures - opens a new route toward large-scale production of a more efficient antigenic compound to be used as a vaccination tool or as an adjuvant, and also represents a top-quality biomaterial to be further modified for biotechnological purposes.
eng
Bugli, F., Caprettini, V., Cacaci, M., Martini, C., Paroni Sterbini, F., Torelli, R., Della Longa, S., Papi, M., Palmieri, V., Giardina, B., Posteraro, B., Sanguinetti, M., Arcovito, A., Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein, <>, 2014; 9 (Maggio): 2727-2739. [doi:10.2147/IJN.S60014] [http://hdl.handle.net/10807/58553]
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10807/58553
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