2.7 Fed-batch cultivation and purification of PCP antigen proteins
The fed-batch culture was carried out in a 5 L bioreactor (Applikon, Holland) with a working volume of 2 L. After overnight activation in LB medium, 200 mL E. coli seed solution was transferred to 1.8 L TBSB medium. Throughout the fermentation process, the dissolved oxygen was maintained at 30% (v/v). pH was controlled at 7.0, and the stirring speed was set at 400-1000 rpm. The temperature was first maintained at 37 °C for 2 h and then changed to 25 °C after IPTG addition. The induction conditions refer to the optimum level determined in MWP. To avoid starvation, a feed medium (50 g/L yeast extract, 714 g/L glycerin, and 25 g/L (NH4)2SO4) was added with a 3.5 mL/h/L feeding rate after 12 h of inoculation. The antigen protein was purified using an AKTA purifier system (GE, Sweden) and a HisTrap HP affinity column. The purity of the purified protein was then determined by SDS-PAGE analysis.