Integrated strategy for efficient production and direct purification of EGFP from unclarified feedstocks

Efficient recovery of recombinant proteins from unclarified feedstocks remains a major challenge in bioprocessing. Enhanced green fluorescent protein (EGFP) is widely used as a model protein, but its purification typically involves multiple clarification steps, increasing processing time and cost. This study presents an integrated strategy to optimize both upstream cultivation and downstream purification for EGFP production and recovery. A two-level, three-factor fractional factorial design (FFD) was employed to optimize environmental variables, identifying optimal conditions at pH 7.0, 26 °C, and 1.75 h of induction time. The optimized culture was scaled up in a 5 L bioreactor, yielding 3.66 mg/mL of EGFP and 36.86 mg/mL of total protein from 50 % (w/v) disrupted E. coli cells. Stirred fluidized bed adsorption (SFBA) using STREAMLINE DEAE adsorbent enabled direct purification from unclarified feedstock. At a linear velocity of 100 cm/h and a stirring speed of 100 rpm, the SFBA process achieved 96.3 % recovery, a 2.9-fold purification factor, and a productivity of 2.82 × 10⁻² mg/mL/min—surpassing previously reported methods. These results demonstrate an efficient, scalable purification approach for EGFP recovery directly from crude lysates, eliminating the need for centrifugation or filtration.