Production of recombinant bone morphogenetic protein‐2 in Escherichia coli : process optimization in shake flask and bioreactor cultures using statistical experimental design

Abstract BACKGROUND This study aimed to enhance production of bone morphogenetic protein‐2 (BMP‐2), a therapeutic agent for treatments of bone‐associated diseases, from engineered Escherichia coli BL21 (DE3) cultures. A central composite design (CCD) was initially applied to evaluate effects of process variables in a shake flask – time, inoculum size, and lactose concentration – on BMP‐2 production, and determine their optimal levels. With the obtained optimal shake flask condition from the first CCD, another CCD was then performed to reveal the effects of process variables in the bioreactor – aeration rate and agitation speed – and suggest their optimal levels. RESULTS Toward maximizing BMP ‐2 production, the desirability analysis based on the resulting statistical models from the first CCD suggested optimal levels of those flask variables, one of which – ~ 9 h, 7% inoculum size, and 3 g L −1 lactose concentration – gave the highest BMP ‐2 concentration and BMP ‐2:dry cell weight ( DCW ) ratio of 192.5 mg L −1 and 118.4 mg g −1 in subsequent experimental validation. From the desirability analysis with the model from the subsequent CCD , only ~1 vvm and 250 rpm were suggested as their optimal levels, experimentally providing the highest BMP ‐2 production; 195.9 mg L −1 BMP ‐2 concentration and 135.9 mg g −1 BMP ‐2: DCW ratio in bioreactor cultures. CONCLUSION Compared to non‐optimized cultures, 23–27% and 36–44% increases in BMP‐2 concentration and BMP‐2:DCW ratio were achieved with the suggested optimal culture conditions. All the experimental data fell within the statistical 95% prediction intervals, ensuring validity of the obtained model predictions. © 2025 Society of Chemical Industry (SCI).