TY - JOUR
T1 - Impact of physicochemical parameters on in vitro assembly and disassembly kinetics of recombinant triple-layered rotavirus-like particles
AU - Mellado, Maria Cândida Maia
AU - Mena, Jimmy A.
AU - Lopes, António
AU - Ramírez, Octavio T.
AU - Carrondo, Manuel J.T.
AU - Palomares, Laura A.
AU - Alves, Paula M.
PY - 2009/11/1
Y1 - 2009/11/1
N2 - Virus-like particles constitute potentially relevant vaccine candidates. Nevertheless, their behavior in vitro and assembly process needs to be understood in order to improve their yield and quality. In this study we aimed at addressing these issues and for that purpose triple- and double-layered rotavirus-like particles (TLP 2/6/7 and DLP 2/6, respectively) size and zeta potential were measured using dynamic light scattering at different physicochemical conditions, namely pH, ionic strength, and temperature. Both TLP and DLP were stable within a pH range of 3-7 and at 5-258C. Aggregation occurred at 35-458C and their disassembly became evident at 658C. The isoelectric points of TLP and DLP were 3.0 and 3.8, respectively. In vitro kinetics of TLP disassembly was monitored. Ionic strength, temperature, and the chelating agent employed determined disassembly kinetics. Glycerol (10%) stabilized TLP by preventing its disassembly. Disassembled TLP was able to reassemble by dialysis at high calcium conditions. VP7 monomers were added to DLP in the presence of calcium to follow in vitro TLP assembly kinetics; its assembly rate being mostly affected by pH. Finally, DLP and TLP were found to coexist under certain conditions as determined from all reaction products analyzed by capillary electrophoresis. Overall, these results contribute to the design of new strategies for the improvement of TLP yield and quality by reducing the VP7 detachment from TLP.
AB - Virus-like particles constitute potentially relevant vaccine candidates. Nevertheless, their behavior in vitro and assembly process needs to be understood in order to improve their yield and quality. In this study we aimed at addressing these issues and for that purpose triple- and double-layered rotavirus-like particles (TLP 2/6/7 and DLP 2/6, respectively) size and zeta potential were measured using dynamic light scattering at different physicochemical conditions, namely pH, ionic strength, and temperature. Both TLP and DLP were stable within a pH range of 3-7 and at 5-258C. Aggregation occurred at 35-458C and their disassembly became evident at 658C. The isoelectric points of TLP and DLP were 3.0 and 3.8, respectively. In vitro kinetics of TLP disassembly was monitored. Ionic strength, temperature, and the chelating agent employed determined disassembly kinetics. Glycerol (10%) stabilized TLP by preventing its disassembly. Disassembled TLP was able to reassemble by dialysis at high calcium conditions. VP7 monomers were added to DLP in the presence of calcium to follow in vitro TLP assembly kinetics; its assembly rate being mostly affected by pH. Finally, DLP and TLP were found to coexist under certain conditions as determined from all reaction products analyzed by capillary electrophoresis. Overall, these results contribute to the design of new strategies for the improvement of TLP yield and quality by reducing the VP7 detachment from TLP.
KW - Assembly kinetics
KW - Capillary zone electrophoresis
KW - Disassembly kinetics
KW - Light scattering
KW - Rotavirus-like particles
UR - http://www.scopus.com/inward/record.url?scp=70350492883&partnerID=8YFLogxK
U2 - 10.1002/bit.22430
DO - 10.1002/bit.22430
M3 - Article
C2 - 19623564
SN - 0006-3592
VL - 104
SP - 674
EP - 686
JO - Biotechnology and Bioengineering
JF - Biotechnology and Bioengineering
IS - 4
ER -