Fluorescent Protein Production, Purification, and Coupling to Microspheres

Area of Science:

• Biotechnology
• Molecular Biology
• Analytical Chemistry

Background:

• Fluorescent proteins (FPs) are vital tools in biological research, with excitation spectra spanning UV to NIR.
• Conventional flow cytometry requires careful filter selection to manage spectral overlap from broad FP emission spectra.
• Multi-FP experiments necessitate single-color controls, which can be inconvenient and costly, especially for systems like the confetti system.

Purpose of the Study:

• To present a simplified alternative for using fluorescent proteins in flow cytometry.
• To describe a method for producing and purifying FPs and coupling them to microspheres for use as stable reagents.
• To provide protocols for evaluating the fluorescent properties of these microsphere-coupled FPs.

Main Methods:

• Expression and purification of recombinant fluorescent proteins (e.g., mPlum) in Escherichia coli.
• Covalent coupling of purified FPs to carboxylate polystyrene microspheres.
• Evaluation of fluorescent properties, including comparison of cell-bound and bead-bound fluorescence, and spectral signature analysis.

Main Results:

• Successfully produced and purified FPs, and coupled them to microspheres.
• The resulting microsphere reagents are stable, can be stored for extended periods, and simplify spectral compensation.
• Protocols for characterization and comparison of spectral properties were established.

Conclusions:

• Coupling purified fluorescent proteins to microspheres offers a convenient and cost-effective alternative to traditional single-color controls in flow cytometry.
• These FP-coupled microspheres serve as stable, ready-to-use reagents that simplify instrument setup and data analysis.
• The described methods enable the generation of reliable fluorescent standards for various biological applications.