## 3 Results
### Cultivation of Recombinant Protein-Producing Cells in Media Containing PFCs
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**PFCs**} & \multicolumn{6}{c}{**Concentration (\% r/v)**} \\ \cline{2-7}  & **0.3** & **0.5** & **1** & **2** & **3** & **5** \\ \hline Perfluorohexane & 389.4 \(\pm\) 19.2 & 410.7 \(\pm\) 19.8 & 384.8 \(\pm\) 18.9 & 325.6 \(\pm\) 18.0 & 335.0 \(\pm\) 16.2 & 271.9 \(\pm\) 13.2 \\ Perfluorodecalin & 334.4 \(\pm\) 16.1 & 339.9 \(\pm\) 16.8 & 341.7 \(\pm\) 16.8 & 360.1 \(\pm\) 16.9 & 337.0 \(\pm\) 16.5 & 325.8 \(\pm\) 15.3 \\ Polyether I & 373.9 \(\pm\) 18.3 & 384.2 \(\pm\) 18.7 & 396.7 \(\pm\) 19.6 & 337.7 \(\pm\) 19.2 & 371.6 \(\pm\) 18.2 & 350.3 \(\pm\) 16.5 \\ Polyether II & 361.7 \(\pm\) 18.0 & 441.1 \(\pm\) 21.2 & 463.5 \(\pm\) 22.3 & 383.5 \(\pm\) 20.7 & 380.0 \(\pm\) 18.6 & 308.2 \(\pm\) 14.9 \\ \hline \hline \end{tabular}  
* Total His\({}_{6}\)-OPH-activity of cells cultivated in the absence of PFCs (control) was 325.6 \(\pm\) 15.3 U/L.  
\end{table} 
Table 3: Total His\({}_{6}\)-OPH activity (U/L), accumulated in the _E. coli_ SG13009[pREP4] cells in the presence of various PFCs in the culture medium *. The culture medium thus obtained was used for the accumulation of the next batch cultivation of His\({}_{6}\)-OPH-containing cells (Figures 2 and 3). This procedure was repeated five times. For control, the _E. coli_ SG13009[pREP4] cells were cultivated in TYE culture medium, where 1/4 of the yeast extract was replaced with disintegrated biomass accumulated in the absence of PFH. The total level of enzyme activity accumulated in the second stage of His\({}_{6}\)-OPH biosynthesis, when \"recycled\" PFH was used instead of the \"pure\" one, was little lower than that obtained from the first stage. During the following three cycles of using the \"recycled\" PFH, the amount of accumulated biomass remained practically the same, whereas the level of His\({}_{6}\)-OPH activity slightly decreased. The latter was probably due to the gradual loss of PFH in the \"recycling\" process. This loss, though inevitable due to the high volatility of PFH, was however not significant, as can be concluded from the results of cultivation. Electrophoretic analysis under non-denaturing (Figure 4a) and denaturing conditions (Figure 4b) was performed for His\({}_{6}\)-OPH protein isolated from the biomass of _E. coli_ SG13009[pREP4] cells that accumulated during cultivating under standard conditions, as well in case of addition of 0.5 vol. % of PFH, both the pure one and that \"recycled\" from the disintegrated biomass remaining from the previous stage. All of the samples produced similar bands near the 36 kDa marker, which are routinely observed during analysis of purified His\({}_{6}\)-OPH via SDS electrophoresis following the standard technique [32]. There were received lines at the level of the protein marker, with a molecular weight of 36 kDa in all of the samples under denaturing conditions, as standard obtained by analysis of purified His\({}_{6}\)-OPH by using SDS-PAGE in a known manner [32]. It was established that the addition of PFH to the medium for cell cultivation slightly increased the total amount of the His\({}_{6}\)-OPH in the cells, and the amount of soluble protein fraction (Figure 4), which explains the observed increase in His\({}_{6}\)-OPH activity in the cells cultured in the presence of PFH. The PAGE analysis of the same proteins that were isolated from the biomass of _E. coli_ SG13009[pREP4] cells under non-denaturing conditions showed that PFH addition in the medium leads to a noticeable decrease in the share of oligomerization forms of the enzyme (Figure 4a), which as a rule, have less enzymatic activity [33]. Thus, the addition of PFH to culture medium for _E. coli_ SG13009[pREP4] cells increased the yield of soluble and active form of the enzyme His\({}_{6}\)-OPH. Figure 3: Accumulation of the total His\({}_{6}\)-OPH activity in the course of multiple batch re-use of PFH (white bars–control, black bars—cells cultivated in the medium containing 0.5% (\(v/v\)) of PFH).