A scalable suspension insect cell transfection method for production of baculoviruses with low amplification passages
Aline Diniz Cabral,
Felipe Baena Garcia,
Renata Torres da Costa,
Ligia Marinho Pereira Vasconcelos,
Mabel Uehara,
Edmar Silva Santos,
Márcia Aparecida Sperança
Affiliations
Aline Diniz Cabral
Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Campus São Bernardo do Campo, Rua Arcturus, 03 - Jardim Antares, Bloco Delta, Sala 226, Laboratório 107, CEP 09606-070, São Bernardo do Campo, São Paulo, Brazil
Felipe Baena Garcia
Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Campus São Bernardo do Campo, Rua Arcturus, 03 - Jardim Antares, Bloco Delta, Sala 226, Laboratório 107, CEP 09606-070, São Bernardo do Campo, São Paulo, Brazil
Renata Torres da Costa
Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Campus São Bernardo do Campo, Rua Arcturus, 03 - Jardim Antares, Bloco Delta, Sala 226, Laboratório 107, CEP 09606-070, São Bernardo do Campo, São Paulo, Brazil
Ligia Marinho Pereira Vasconcelos
Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Campus São Bernardo do Campo, Rua Arcturus, 03 - Jardim Antares, Bloco Delta, Sala 226, Laboratório 107, CEP 09606-070, São Bernardo do Campo, São Paulo, Brazil
Mabel Uehara
Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Campus São Bernardo do Campo, Rua Arcturus, 03 - Jardim Antares, Bloco Delta, Sala 226, Laboratório 107, CEP 09606-070, São Bernardo do Campo, São Paulo, Brazil
Edmar Silva Santos
Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Campus São Bernardo do Campo, Rua Arcturus, 03 - Jardim Antares, Bloco Delta, Sala 226, Laboratório 107, CEP 09606-070, São Bernardo do Campo, São Paulo, Brazil
Márcia Aparecida Sperança
Corresponding author.; Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Campus São Bernardo do Campo, Rua Arcturus, 03 - Jardim Antares, Bloco Delta, Sala 226, Laboratório 107, CEP 09606-070, São Bernardo do Campo, São Paulo, Brazil
Baculovirus expression vector systems (BEVS) have been widely used for production of recombinant proteins in insect cells. However, baculoviruses superinfection and repeated passages originate defective interfering particle (DIP) mutants, which is a limitation to a continuous large-scale production. Accordingly, a classical chemical transfection method performed on monolayer of Spodoptera frugiperda insect cells (Sf9) was modified to produce recombinant baculoviruses with high efficiency. Modifications consist to transfect exponentially growing cells in suspension after concentration by tenfold through centrifugation. Ten different constructions of recombinant baculoviruses with insert varying in size from 180 bp to 2,395 bp, were obtained through employment of the Bac-to-Bac expression system (ThermoFisher/Invitrogen). The transfection efficiency of the modified protocol varied from 45 to 57%, independent of the insert size, while classical method present transfection efficiency of 2 to 20%. After transfection of 6 × 106 cells, the recombinant baculoviruses titer obtained with modified method was about 2 × 107 pfu/ mL in a total volume of 12 mL, which is scalable to 24 liters of 1 × 108 pfu/ mL, after only two amplification rounds, contributing to improve large scale heterologous protein production in insect cells, with low amplification passages.
