Development of a defined medium for heterologous expression in Leishmania tarentolae.

A defined medium is essential for studying nutritional requirements of microorganisms and for selective supplementation of necessary substances. Hemin is an essential ingredient for growth of Leishmania tarentolae, but tends to precipitate in aqueous solutions without further stabilization. These aggregates disturb the measurement of the optical density or the cell density and the following downstream processing. Therefore, we were looking for stabilizing substances and established a PEG-hemin-solution, which avoided flocculation and allowed the cultivation of L. tarentolae in a medium, which we termed SFP(II) medium. With addition of RNA from Saccharomyces cerevisiae to SFP(II) medium the SFP(III) medium was established. In this medium, the specific cell division rate was increased (0.103 h(-1)) and stable for longer periods of time. The evaluation of the SFP(III) medium was done in shaker flasks by successful expression and segregation of the SAG2 protein, one of the main surface antigens of Toxoplasma gondii. With establishment and evaluation of this defined medium, the status of the Leishmania tarentolae expression system as an alternative to commonly used cell cultures is supported.

Characterization of the growth behavior of Leishmania tarentolae: a new expression system for recombinant proteins.

Biotechnological production of recombinant proteins for human therapy requires a cultivation of the host organism in a nutrient medium free of animal substances. Therefore, various nutrient media for the new expression system Leishmania tarentolae were developed and examined according to their cultivation conditions as static suspension culture and agitated culture. Investigations resulted in the development of a serum-free but hemin containing medium, based on yeast extract and buffer salts. Here we report that a high and stable specific growth rate of 0.103 h(-1) and a maximal cell density of 1 x 10(9) cells ml(-1) is obtained in an alternative medium, the YE-medium. For the newly developed medium, the successful expression of enhanced green fluorescent protein and the adaptation of the cultivation from the agitated culture to the bioreactor could be shown. Furthermore, an analytical method for detection of the essential, organic iron source hemin was established. The consumption of hemin was monitored because hemin is a potentially important process parameter for bioprocess control. With knowledge of these results, an improved expression system is available as an alternative to commonly used cell cultures for the production of recombinant proteins.