Making GFP count: a validated framework for absolute protein quantification in precision fermentation

Abstract Precise quantification of recombinant proteins is essential for assessing and comparing expression efficiency and optimizing production processes. Fluorescent proteins have emerged as powerful tools for real-time monitoring of gene expression and protein tracking. However, standardized and validated methods for their quantification, particularly for the widely used green fluorescent protein, remain limited. To date, no universally adopted protocol has emerged. This study presents a high-throughput method for the quantification of recombinantly produced Emerald Green Fluorescent Protein (EmGFP) based on direct fluorescence measurements of the cell suspension while quantifying and integrating potential effects of signal attenuation. The workflow uses solely standard laboratory equipment, ensuring broad accessibility and easy implementation. Moreover, in-house EmGFP standard preparation and quantification is described. The method was validated according to FDA guidelines “Analytical Procedures and Methods Validation for Drugs and Biologics,” addressing the requirements of linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, and recovery rate. Investigation was conducted using Escherichia coli BL21 cells expressing EmGFP, widely available sodium fluorescein as a chemical standard, commercial GFP, and an in-house EmGFP standard. A robust correlation (linear fitting, R 2 0.96) of the EmGFP concentration and relative fluorescence units (RFU) was established, enabling efficient and high-throughput fluorescence quantification using a standardized workflow in a microtiter-based format suitable for the application in comparative studies across different expression constructs, conditions, and scales. By enabling absolute quantification of fluorescent proteins, this method supports both real-time bioprocess optimization and broader applications in protein production research.