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

Area of Science:

• Biotechnology
• Protein Expression and Quantification

Background:

• Accurate quantification of recombinant proteins is crucial for optimizing production and assessing expression efficiency.
• Standardized methods for quantifying fluorescent proteins, like green fluorescent protein, are limited, hindering comparative studies.

Purpose of the Study:

• To develop and validate a high-throughput, accessible method for quantifying recombinantly produced Emerald Green Fluorescent Protein (EmGFP).
• To establish a standardized workflow for absolute quantification of fluorescent proteins using direct fluorescence measurements and accounting for signal attenuation.

Main Methods:

• Developed a high-throughput workflow for EmGFP quantification using direct fluorescence measurements of cell suspensions.
• Integrated signal attenuation correction and validated the method using FDA guidelines, including linearity, LOD, LOQ, precision, accuracy, and recovery.
• Utilized standard laboratory equipment, Escherichia coli BL21 cells, sodium fluorescein, commercial GFP, and an in-house EmGFP standard.

Main Results:

• Established a robust correlation (R² = 0.96) between EmGFP concentration and relative fluorescence units (RFU) in a microtiter-based format.
• Demonstrated the method's suitability for high-throughput, comparative studies across various expression constructs and conditions.
• Successfully quantified in-house EmGFP standards and validated the method's accuracy and precision.

Conclusions:

• The presented method offers an accessible, standardized, and high-throughput approach for absolute quantification of fluorescent proteins.
• This workflow facilitates real-time bioprocess optimization and supports broader research in protein production.
• Enables precise assessment of expression efficiency and facilitates comparisons across different experimental scales and conditions.