A scalable suspension insect cell transfection method for production of baculoviruses with low amplification passages

Abstract

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.

Keywords: Baculovirus; Insect cell; Sf9; Transfection.

Graphical abstract

Fig. 1

Schematic representation of recombinant bacmid construction through transposition process. Tn7R and Tn7L: transposase recognition sequence, MiniAtt-Tn7: transposase sequence target; TET, GENT, AMP, KAN: tetracycline, gentamycin, ampicillin and kanamycin resistance marker. (Adapted from: Invitrogen - Bac-to-Bac^Ⓡ Baculovirus Expression System User Guide, 2013).

Fig. 2

Microscopic analysis of Sf9 insect cells stained with trypan blue after transfection with Mayaro Nsp2 recombinant baculovirus DNA. Non-transfected (-) and transfected (+) Sf9 cells after 24, 48, 72 and 96 h post transfection experiment. Microscopic magnification of each picture is indicated in the figure.

Fig. 3

Microscopic analysis of Sf9 insect cells before and after transfection with Mayaro Nsp2 recombinant baculovirus with 40x magnification. (a) Non-infected control, (b) 24 h post transfection. (c) 48 h post transfection; black arrows show non-viable cells stained with trypan blue (d, e, f) 48 h post transfection; white arrow indicates cellular debris and the black arrow indicates the formation of vacuolar structures next to the inner nuclear membrane of the cell. (g, h, i) 72 h post transfection.