Analysis of Fermentation Process for Scfv Production by Proteomics

During the production phase of the culture for the foreign protein production by recombinant Pichia pastoris, the control of methanol feeding is important because methanol is used not only for protein production but also for the growth of the strain.  The starvation of the carbon source inhibits the growth of the strain and the production of foreign protein. Furthermore, excess methanol concentration in culture broth severely affected the metabolism in the strain. Therefore, we constructed the methanol feeding control system with the aid of semiconductor gas sensor. Using this system, we increased the production amount of a model foreign protein, e.g. single-chain Fv antibody (scFv). As a result of the fed-batch culture by a 2L jar-fermenter, we obtained 73 g/L of the dry cell weight (DCW) and 1.5g/L of scFv concentration, which is 5 times higher than the upper limit of the product concentration obtained in the flask cultivation. The scFv production started soon after the start up of methanol feeding operation. The production continued around 100 hours and the production rate was kept almost constant. Then, we analyzed the cell in the growth and production phase with two-dimensional electrophoresis (2-DE). The images obtained from the cells in different phases are quite different, which suggested that the expression protein of the cells seriously changed with the phase of cultivation.