Purification of recombinant rotavirus VP7 glycoprotein for the study of in vitro rotavirus-like particles assembly.
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 1x10(6)cellsmL(-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 0.1plaque forming unit (pfu)cell(-1) and time of harvest of 80h 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 MW and apparent MW were determined as 31.6 and 39kDa, respectively, confirming the efficacy of the proposed purification strategy that now enables RLPs assembly studies.
Mellado
Maria Candida M
MC
ITQB-UNL/IBET, Apartado 12, 2781-901 Oeiras, Portugal.
Peixoto
Cristina
C
Cruz
Pedro E
PE
Carrondo
Manuel J T
MJ
Alves
Paula M
PM
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015153:Blotting, Western; D036022:Capsid Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D019065:Virus Assembly / Q000502:physiology*
D000956:Antigens, Viral; D036022:Capsid Proteins; D011994:Recombinant Proteins; C050779:VP7 protein, Rotavirus