RESUMO
OBJECTIVE: To develop a method for the efficient assembly of viral or multimeric proteins into virus-like particles (VLP) or other macro structures. RESULTS: Protein monomers were assembled by eliminating calcium ions through precipitation. The model protein, rotavirus VP6, assembled into stable, long nanotubes with better quality than the assemblies obtained directly from cell culture. Nanotube length was directly proportional to the initial concentration of VP6 monomers, in accordance with the classic nucleation theory of capsid assembly. The quality of the obtained assemblies was confirmed when the nanotubes were functionalized with metals, yielding unique nanobiomaterials. Assembly efficiency was improved in comparison with other previously proposed methods. CONCLUSIONS: The novel method presented here is simpler and faster than other reported methods for the assembly and disassembly of viral proteins, a step needed for most applications.
Assuntos
Antígenos Virais/química , Antígenos Virais/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Rotavirus/metabolismo , Cálcio/química , Precipitação Química , Nanotubos/química , Multimerização ProteicaRESUMO
Rotavirus VP6 self-assembles into high order macrostructures useful as novel scaffolds for the construction of multifunctional hybrid nanobiomaterials. This application requires large quantities of high quality pure material with strict structural consistency. Strategies for obtaining high quality recombinant VP6 and different characterization techniques are explored and compared in this work. VP6 was expressed in the insect cell-baculovirus system. VP6 assemblies were selectively purified utilizing an ion exchange and size exclusion (SE) chromatography. Purification steps were monitored and characterized by dynamic light scattering (DLS), ELISA, SDS-PAGE, HPLC and Western blot. DLS showed that the initial ultrafiltration step removed small particles, the intermediate anion exchange chromatographic step completely removed the baculovirus, whereas the final size exclusion chromatography permitted the selective recovery of correctly assembled VP6 nanotubes and discrimination of non-assembled VP6, as confirmed by transmission electron microscopy. VP6 assembled into tubular structures with diameter of 75 nm and several nanometers in length. The purification yield was 20% of multimeric assemblies with a purity >98%. The resulting material was suitable for the production of functionalized hybrid nanobiomaterials through in situ synthesis of metallic nanoparticles.