P52: Optimization of enterokinase production in MTT1-ENT Tetrahymena thermophila producing enterokinase via a cadmium-induced promoter

Tetrahymena thermophila, a ciliated protozoan, has been used in cellular and molecular biological experiments and in-vivo genomic expression studies. Its potential use for industrial-scale protein production has recently been recognized, but has not yet been fully investigated. Using T. thermophila for protein production is advantageous; it requires inexpensive medium, grows with a fast doubling rate of 4 hours, and has a well-studied genome. This work uses a novel line of T. thermophila,  MTT1-ENT, which has an extra chromosome-like plasmid containing the gene encoding bovine enterokinase (ENT) under the control of a cadmium-induced promoter. Bovine enterokinase is similar in size and complexity to a malaria vaccine, which MTT1 can produce. NEFF medium, containing yeast extract, glucose, and proteose peptone, is often used for T. thermophila growth.

This work investigates the optimal medium composition and the effect of using different peptones and nutrients on the ENT production. Using Design of Experiment (DOE) methods in a novel 48-well mini-bioreactor, with each well equipped where pH, dissolved oxygen, and cell concentration can be monitored in real-time. The effect of medium composition on amino acid uptake and ENT specific activity is also examined. Initial findings show that higher glucose concentrations result in greater activity of the produced ENT, and that MTT1-ENT cells show a 50% increase in specific activity and 37% increase in cell concentration when growing in casein peptone compared to proteose peptone. The results obtained for MTT1-ENT protein production should contribute to make MTT1 T. thermophila an accessible line for future protein applications.