title: Strategies for the purification and characterization of protein scaffolds for the production of hybrid nanobiomaterials.
abstract: 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.
authors.affiliation: Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico. germanpv@ibt.unam.mx
authors.forename: Germán
authors.initials: G
authors.lastname: Plascencia-Villa
authors.forename: Jimmy A
authors.initials: JA
authors.lastname: Mena
authors.forename: Ricardo M
authors.initials: RM
authors.lastname: Castro-Acosta
authors.forename: Julio César
authors.initials: JC
authors.lastname: Fabián
authors.forename: Octavio T
authors.initials: OT
authors.lastname: Ramírez
authors.forename: Laura A
authors.initials: LA
authors.lastname: Palomares
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D001709:Biotechnology / Q000295:instrumentation / Q000379:methods*; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D002850:Chromatography, Gel / Q000379:methods; D046529:Microscopy, Electron, Transmission; D043942:Nanotubes / Q000737:chemistry* / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000821:virology
chemical_list: D000956:Antigens, Viral; D036022:Capsid Proteins; D011994:Recombinant Proteins; C061332:VP6 protein, Rotavirus