Analysis of the factors influencing optimal recombinant protein expression

Several diverse types of proteins have been expressed using the baculovirus-infected insect cells.

The expression level is depending on the nature of the protein being expressed (Kost et al., 2005).

GFP is a highly stable protein and cannot be easily degraded by proteases in intact cells (Li et al., 1998). In this study, experiments were performed to analyse the correlation between cell viability and maximal protein expression. Additionally, the effects of different MOIs on protein expression in the presence of different culture conditions were also observed. Low MOI strategy was used to analyse the recombinant GFP expression from different time points post-infection and its dependency on various cell kinetic parameters.

4.2.1 Protein expression analysis in serum-free culture condition

For serum-free culture conditions (Fig.3.7), the cell viability increased for 24 h.p.i for all MOIs (0.08, 0.4, and 0.8) tested, which was unexpected. An explanation for this increase, a presence of defective virus particles could be speculated. Another possible reason could be an unsynchronized infection by amplified baculovirus due to low MOIs, and the cell population could lack the uniformity of metabolic status (Contreras-Gómez et al., 2014). During low MOI infection, a small number of cells get infected while others continue to divide. That means, when considering the whole culture, the energy and nutrition requirements varied greatly for each cell during prolonged infection. On the contrary, during high MOI infection, there could be variation in energy and nutrition requirements initially but after a short time period, nearly all cells will be infected and could have uniform metabolic status. Then from 24 h.p.i to 48 h.p.i, the viability starts to decrease for all MOIs. However, there was no detection of GFP bands on western blots during this period for all MOIs. After 48 h.p.i, there was a significant increase in the protein expression signal for MOIs 0.4 and 0.8, but the cell viability did not decrease any further. Hence, there was no correlation observed between cell viability and protein expression. On the other hand, the protein expression signal was proportional to MOIs and infection time. However, it is clear that the cell viability of uninfected cells was also decreased continuously, and during the experiment, there was also observed a low level of white material in all spinner flasks, including the uninfected spinner flask. These materials may be dead cells or cell debris. An explanation could be that cells died through shear stress. Although, cell media contains the shear protective non-ionic copolymer surfactant Pluronic F-68. There is also a possibility that cells might have been stressed due to cell scrapping procedure within the adherent flasks, but cell conditions could not be properly recognized through an inverted microscope observation. Further transfer of these cells to spinner

52 flasks could have made them even more vulnerable. Additionally, there was no indication of any biological contamination observed.

4.2.2 Protein expression analysis in serum-rich culture condition

GFP is an intracellular protein distributed all over the cytoplasm and nucleus in transfected cells (Leiva et al., 1976). GFP is relatively stable and not easily degraded by the protases (Gotoh et al., 2001), but due to increasing cell lysis, it is released into the medium if the time of infection is prolonged (Sander et al., 2007). With an addition of 2% FCS, it was observed that cell doubling time was reduced during adherent culture. Unlike the serum-free culture conditions, cells seemed in good shape under the influence of serum in spinner flasks. FCS contains many growth stimulating factors and protects the cells from sheer stress. Thus, serum-rich conditions can be favorable for cell growth. Cell viability of uninfected cells also remained more than 90 percent for five days as seen in Fig. 3.8. But for virus-infected cells, cell viability varies for all three different MOIs. Sander et al. (2007) mentioned in their research that Jain et al. (1991) have found a 50 % decrease in cell viability at the peak production of antistasin when Sf9 cells were infected with the recombinant virus at MOI = 0.1. However, in this study when comparing figures 3.8 and 3.9 (D), there was no correlation between a particular drop of viability and the time of peak expression of GFP when cultures were infected at different MOIs. Although the time point of maximal GFP expression for the different MOIs was similar with 72 h.p.i, the protein expression with MOI = 0.08 showed the highest signal of expression, higher than the cultures infected at MOI = 0.4 and MOI =0.8. Additionally, the peak protein expression at MOI = 0.4 and MOI = 0.8 were nearly similar. Lowest MOI infection has the highest protein expression. However, this inverse relation between MOI and protein expression did not remain valid MOI=0.4 and MOI=0.8. Under the conditions, when the infection is started with MOI less than 1, the accumulation of maximum protein is affected by the dynamic influence of the virus propagation, continuing cell division, and the nutritional condition of the medium. Therefore, the correlation between MOI and time of maximum protein activity can be very complex (Yang et al., 1996).

In order to investigate the optimal protein expression conditions, other parameters like changes in viable cell density and the average cell diameter, obtained from the automated cell counter, were compared for each sample of these three MOIs infected cells (data not shown). Before infection, all the spinner flasks have an initial viable cell density of 1.0 x 10⁶ cells/ml. It was observed that culture infected with MOI = 0.08 and 0.4 showed a continuous increase of viable cell density and reached the peak viable cell density of 1.5 x 10⁶ cells/ml and 1.7 x 10⁶ cells/ml at 96 h.p.i, respectively. That means the viable cell density increases for both MOIs beyond the peak of protein expression, which was 72 hours for both MOIs. This increase in viable cell numbers at 96 h.p.i.

can arise the question of potency and effectivity of the virus. Because theoretically, as the infection progresses, the numbers of budded virus progeny should increase which then can infect large numbers of non-infected cells. However, viable cell density of the culture infected with MOI = 0.8 follows the pattern of protein expression profile. The viable cell density of MOI= 0.8 was maximum of 1.5 x 10⁶ cells/ml at 72 h.p.i. After that, it decreases to 1.4 x 10⁶ cells/ml at 96 h.p.i as the protein expression during this time point also decreases. But, only one condition and one experiment are insufficient to generalize the hypothesis that a peak of protein expression can be predicted from the peak of viable cell density of the culture. Several experiments regarding this question are needed.

53 Another parameter mentioned, the changes in average cell diameter was also analysed. As described in the introduction section, baculovirus-infected cells increase in cell size due to the accumulations of viral DNA post-infection. Moreover, increased cell size also indicates the progression of the infection. The larger cells will have a higher potential to increase recombinant protein synthesis. Palomares et al. (2001) found a direct correlation between the maximum average cell diameter and the total protein production when Sf9 cells were infected with a baculovirus encoding VP8 protein. However, in this study, such a correlation was not observed. Initial average cell diameters at 0 h.p.i of Sf9 cells, infected with amplified baculovirus at MOI 0.08, 0.4, 0.8 were 12.4 μm, 12.1 μm and 12.3 μm, respectively. The maximum average diameters of infected Sf-9 cells were observed at 96 h.p.i for the samples from each MOI, which were 15.2 μm, 15.4 μm, and 15.9 μm, respectively. The time point of peak protein expression did not correlate with peak cell diameter, indicating the increase in cell size resulted from viral protein synthesis rather than recombinant protein expression (Palomares et al., 2001).

4.2.3 Protein degradation

Apart from the finding that cell kinetic parameters did not correlate with the protein expression profile, there was also a significant amount of protein degradation observed. Multiple degradation bands are present as can be seen in Fig.3.9 and 3.10 of analysed PVDF membranes. Because of the lytic nature of the baculovirus expression system, intracellular and extracellular proteases are in contact with recombinant protein. Proteases can be produced by the insect cells as a defense mechanism of stress, caused by baculovirus (Ikonomou et al., 2003). The baculovirus vector can also have the potential to increase protease concentration (Licari et al., 1991). Lysosomal proteases can be released upon cell lysis as the infection progresses (Ikonomou et al., 2003). During the cellular defense mechanism, there is a possibility that cellular proteases may target the recombinant protein for the proteolytic action. There may also occur direct degradative action from the baculovirus (Licari et al., 1991). Many approaches have been documented to reduce the protein degradation problems for the baculovirus expression system. One approach can be the use of earlier promotors rather than late promoters like p10 or polh, which are active when cells undergo lysis and release proteases. The second approach can be deletion of genes from the baculovirus construct such as chiA and v-cath, which encode viral enzymes chitinase and cathepsin, respectively. These viral enzymes are accountable for cell lysis, and they are not required for replication and recombinant protein expression. Therefore, using these constructs lysis of insect cells can be prevented, and protein degradation can be reduced. Another application is the addition of the protease inhibitor directly into the culture supernatant of virus-infected cells or in the cell culture media (Ikonomou et al., 2003; Contreras-Gómez et al., 2014).

The expression level of recombinant protein was significantly lower in serum-containing medium compare to serum-free conditions in the presence of DMSO, as shown in figure 3.10 (D). The cell viability of serum-free and serum-containing cultures decreased rapidly, that was because of the DMSO effect rather than baculovirus infection. Usage of serum increase the cost as well as makes

54 purification of the protein more difficult. Serum components contain various growth factors for cell growth and proliferation, along with many other active proteins. A serum can be favorable for cell growth and protein expression but might cause an inhibitory effect on recombinant protein expression in the presence of DMSO. However, both in FCS containing and serum-free conditions, there was the degradation of recombinant protein observed with the addition of DMSO, as can be seen in analysed PVDF membrane images in figure 3.10. Therefore, it is clear that addition of DMSO has not any effect on minimizing the protein degradation either in the presence or absent of FCS. Hom and Volkman (1998) have found that, preparation of protein in sample buffer for SDS-PAGE may cause the protein degradation effect. In their research, Sf-21 cells were infected with AcMNPV virus and claimed that v-cath enzyme, which can be responsible for protein degradation, was chemically activated by the SDS sample buffer. Hom and Volkman (1998) recommended the use of E-64 or a similar cysteine protease inhibitor supplementation in the sample of baculovirus-infected cells to prevent the proteolytic action. GFP degradation due to sample buffer could be possible, but other student groups during the cell culture practical course, in the same laboratory, also have performed similar experiments with different MOI. They have not found any putative the degradation bands when using Sf9 cells for similar sample buffer preparation. Thus, for Sf9 cells, degradation effect from sample buffer could not be hypothesized.

To prevent protein degradation from proteases, protease inhibitor cocktail was effective in this study. This protease inhibitor cocktail inhibits the activity of serine, cysteine, and aspartic proteases as well as prevents the protein cleavage by the aminopeptidases enzyme (www.sigmaaldrich.com). As can be seen in figure 3.11 C, there was no detection of protein degradation bands. PI was also toxic for cell growth because it dissolved in the DMSO, which can have a cytotoxic effect. Therefore, a decreased in the cell viability was observed, but otherwise, protease inhibitor causes a favorable effect on the improvement of protein expression signals. It seems that the addition of protease inhibitor might be a cost-effective solution to reduce the proteolytic events for protein production in the baculovirus in the laboratory research.

4.2.4 Defective interfering particles

Another factor, baculovirus defective interfering (DI) particles also have the potential to cause a dramatic effect on the correlation between MOI and protein expression. DI particles contain a part of the baculovirus genome and normal viral proteins. These particles can interfere and compete in the replication process with the intact recombinant virus. They are produced and enriched by serial passaging and infection of cells with higher MOI (Wickham et al., 1991). As mentioned before, low MOI causes unsynchronized infection. Therefore, the cell population cannot remain in a consistent metabolic status. As a result, the creation of DI particles could increase, and the expressed recombinant protein may be exposed to proteases for long times (Contreras-Gómez et al., 2014). For that reason, the expressed recombinant protein can be more prone to degradation.

The unexpected changes observed in the cell viability could be due to the effect of DI particles.

To overcome the issue of DI particles, one should minimize it by plaque purification of the amplified virus before infecting insect cells.

55 If problems of protein degradation and interference of DI particles can be minimized, tracking the changes in the cell viability, cell density and cell size over time could be an effective way to examine the course of infection and recombinant protein expression. By manipulating the experimental conditions until the expected changes of parameters can be established, one could predict the optimal harvest time and the peak of target protein expression in order to achieve optimal protein production with the baculovirus expression system (Sander et al., 2007). It was also recommended to perform all the similar experiments by using only one large gel instead of many small gels for future experiments. Additionally, the comparison of the expressed protein intensities with bacterial GFP lysate or with other similar house proteins as a reference will provide a better analysis of recombinant protein expression.

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