| The use of genetically engineered parasite cells is a new and novel method of producing recombinant therapeutic proteins. Compared to other animal cell systems, parasites are virtually insensitive to shear in suspension culture, greatly simplifying the scale-up into commercial bioreactors. A study was carried out to develop a low protein serum free media for the in vitro growth of the parasite Leishmania chagasi. Issues studied included the evaluation of commercially available basal media, the improvement of key nutrient utilization, and the optimization of physiochemical and cultures parameters for the high-density axenic culture of Leishmania chagasi promastigotes. Using the developed medium, cell densities approaching {dollar}1times10sp8{dollar} cells/mL were achieved in suspension culture. Subsequently, the aforementioned parasite cells were genetically engineered to over-express two exogenous cytoplasmic proteins in order to assess the potential of a Leishmania based recombinant protein expression system. It was demonstrated that a Leishmania expression system is capable of expressing high levels of non-secreted recombinant protein both in small-scale stationary culture and in laboratory-scale suspension culture. A ten-day batch culture of Leishmania produced an active protein yield of 18 mg/L luciferase. |
