Multiparameter screening method for developing optimized red-fluorescent proteins.

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Genetically encoded fluorescent proteins (FPs) are essential tools in cell biology for protein tracking and biosensing.
• Optimizing FPs for specific applications requires tailoring multiple properties beyond simple brightness.
• Current screening methods often focus on limited parameters, hindering the development of high-performance FPs.

Purpose of the Study:

• To develop and validate a multiparameter screening method for optimizing genetically encoded fluorescent proteins (FPs).
• To simultaneously evaluate critical FP properties including fluorescence lifetime, cellular brightness, maturation efficiency, and photostability.
• To enhance the efficiency and scope of FP optimization workflows.

Main Methods:

• A high-throughput primary screen in bacterial colonies assessing fluorescence lifetime and cellular brightness.
• A secondary multiparameter screen in mammalian cells using a novel dual-expression vector, evaluating four critical parameters.
• Automated multiparameter acquisition and cell-based analysis for 96-well plates to increase workflow efficiency.

Main Results:

• The protocol successfully yielded record-bright mScarlet, fast-maturing mScarlet-I, and photostable mScarlet-H variants.
• Demonstrated the effectiveness of simultaneous screening for multiple FP performance parameters.
• Achieved a complete screening round, including sequencing and data processing, within 2 weeks.

Conclusions:

• The developed multiparameter screening protocol significantly improves the optimization of genetically encoded fluorescent proteins.
• This method enables the rapid generation of FPs with tailored properties for diverse cell biology applications.
• The protocol is adaptable for other FP classes and Förster resonance energy transfer (FRET)-based biosensors.