Integrated optimization of EGFP production and direct recovery using stirred fluidized bed adsorption: Effects of culture and stirring conditions

An integrated approach was developed to optimize upstream production and downstream purification of enhanced green fluorescent protein (EGFP) in Escherichia coli BL21. Culture medium components were optimized using a statistical design of experiments, including a two-level Fractional Factorial Design (FFD) to screen key variables and a Central Composite Design (CCD) with Response Surface Methodology (RSM) to refine critical components. The optimal concentrations of glucose (4.215 g/L) and FeSO₄ (0.297 mg/L) resulted in a 333.1 % increase in EGFP production compared to a modified M9 medium. For purification, STREAMLINE DEAE ion-exchange chromatography was implemented in a stirred fluidized bed adsorption (SFBA) system designed to handle unclarified E. coli lysates (50 %, w/v). Stirring speed was optimized across the 0–200 rpm range, with 100–150 rpm providing the highest adsorption efficiency, recovery yield (∼87 %), and protein purity (purification factor ∼2.7). Unlike traditional packed-bed systems, the SFBA process enabled direct capture from crude lysates, improving throughput, reducing processing time, and enhancing scalability. This study demonstrates the effectiveness of coupling culture optimization with hydrodynamic tuning in SFBA for high-yield, clarification-free recovery of EGFP, offering a practical platform for producing and purifying recombinant proteins in industrial bioprocessing.