Purification of recombinant rotavirus VP7 glycoprotein for the study of in vitro rotavirus-like particles assembly

Abstract

Rotavirus VP7 is a glycoprotein that forms the viral capsid outerlayer and is essential to the correct assembly of triple-layered rotavirus-like particles (RLPs). In this work, a novel purification strategy was designed to allow obtaining highly pure monomeric VP7 required for the RLPs in vitro assembly. VP7 production kinetics in baculovirus–insect cells at cell concentration at infection (CCI) of 1 × 10 6 cells mL −1 was compared in terms of VP7/glycoprotein 64 (gp64) ratio at different multiplicity of infection (MOI). The best productivity was achieved at MOI of 0.1 plaque forming unit (pfu) cell −1 and time of harvest of 80 h post-infection. After preliminary clarification steps, the proteins eluted from Concanavalin A were concentrated and loaded onto size exclusion chromatography. The polishing step was anion exchange chromatography with Mono Q. The high resolution of this column resulted in separation of monomers from dimers of VP7. Overall, the purification protocol yielded high level of purity (>90%). Purified VP7 was characterized by MALDI-TOF mass spectrometry and SDS-capillary gel electrophoresis. The M W and apparent M W were determined as 31.6 and 39 kDa, respectively, confirming the efficacy of the proposed purification strategy that now enables RLPs assembly studies.