Tnao38, high five and Sf9--evaluation of host-virus interactions in three different insect cell lines: baculovirus production and recombinant protein expression

Abstract

A comparative analysis of new and established insect cell lines, in regard to process relevant parameters, provide data that can be exploited for designing more robust and effective protein production processes. The baculovirus-insect cell expression system has been efficiently used for the production of heterologous proteins. Three different insect cell lines Tnao38, High Five and Sf9 were compared in terms of virus susceptibility, baculovirus production and product yield of an intra-cellularly (YFP) and extra-cellularly (influenza A virus hemagglutinin)-expressed recombinant protein. The Tnao38 and High Five cell lines exhibited higher (tenfold) susceptibility to baculovirus infection than Sf9 cells, whereas Sf9 cells showed a higher (100-fold) capacity for production of infectious virus particles. Analysis of recombinant protein expression revealed considerably higher product yields in Tnao38 and High Five cells as compared to Sf9 cells, for both model proteins. Overall, the two Trichoplusia ni-derived cell lines, High Five and Tnao38, were significantly more efficient in terms of secreting proteins such as the glycoprotein hemagglutinin of influenza A virus.

Fig. 1

Schematic representation of baculovirus production and titer determination in and on Tnao38, High Five and Sf9 cells. Tnao38, High Five and Sf9 cells were infected with a YFP-encoding baculovirus. Virus generated in each cell line was subsequently titrated on all three cell lines. Titers were determined via endpoint dilution assay

Fig. 2

Analysis of susceptibility and baculovirus production in Tnao38, High Five and Sf9 cells. Tnao38, High Five and Sf9 cells were infected with AcNPV-H1-Cal09-YFP at an MOI of 1, viral titers raised 6 days post infection were evaluated via endpoint dilution assay on all three cell lines. Titer values are given as plaque forming units (pfu)/ml

Fig. 3

Intracellular YFP expression. Tnao38, High Five and Sf9 cells were infected with AcNPV-H1-Cal09-YFP at an MOI of 10. Cells were harvested at indicated time post infection, cellular pellets were lysed and YFP expression was analyzed by fluorescence measurement in a plate reader. Values for relative fluorescence units (RFU) are given as the average from three technical replicas

Fig. 4

Hemagglutinin expression in Tnao38, High Five and Sf9 cells. Tnao38, High Five and Sf9 cells were infected with AcNPV-H1-Cal09-YFP at an MOI of 10. Cells were harvested at indicated time post infection and pellet and supernatant fractions were subjected to Western blot analysis with mouse anti-H1-Cal09 polyclonal serum. M protein standard, 1–5 Pellet/Supernatant fractions at indicated days post infection (dpi), + (positive control): Ni–NTA purified HA (H1-Cal09), − (negative control) unrelated protein