Kinetics of Protein Secretion in Trichoderma Reesei

In order to obtain knowledge of the protein secretion process in filamentous fungi, and to study the rate-limiting steps in protein production, we have developed methodology for measuring the kinetics of protein production and secretion by metabolic labelling in Trichoderma. The synthesis of the 35S- labelled proteins and their transport into the growth medium was monitored by 2D gel electrophoresis and/or immunoprecipitation. The major cellulose of T.
reesei, cellobiohydrolase I (CBHI), was used as a model protein in the studies. To study the protein production kinetics in a controlled physiological state, a chemostat cultivation system was set up for the fungus. In the chemostat cultivations, the intracellular production rate of labelled CBHI and the accumulation rate of the protein into the culture medium was measured at different dilution rates, and the corresponding specific steady-state mRNA levels were determined. With this methodology we have been able to follow the time course of protein synthesis, glycosylation and transport into the medium,
and obtain information on the synthesis vs. secretion capacity of the fungus in relation to growth rate.