Noncytotoxic DsRed derivatives for whole-cell labeling

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Fluorescent proteins (FPs) are essential tools in biomedical research, enabling visualization of cellular processes.
• Cytotoxicity of orange, red, and far-red FPs, often caused by aggregation, limits their application in live cells.
• Improving FP solubility and reducing aggregation are critical for developing safer and more effective labeling agents.

Purpose of the Study:

• To engineer a non-cytotoxic tetrameric fluorescent protein by reducing aggregation.
• To develop novel orange, red, and far-red FP variants with enhanced solubility and reduced cytotoxicity.
• To establish methods for analyzing FP aggregation and cytotoxicity in bacterial and mammalian cells.

Main Methods:

• Surface engineering of DsRed-Express to create the soluble and non-cytotoxic tetrameric FP, DsRed-Express2.
• Directed evolution of DsRed-Express2 to generate new color variants (E2-Orange, E2-Red/Green, E2-Crimson).
• Assays for evaluating FP cytotoxicity in Escherichia coli and HeLa cells, and methods for analyzing FP aggregation.

Main Results:

• DsRed-Express2 was successfully engineered as a highly soluble, tetrameric FP that is non-cytotoxic in both bacterial and mammalian cells.
• Directed evolution produced novel color variants (E2-Orange, E2-Red/Green, E2-Crimson) derived from DsRed-Express2.
• The developed methods allow for effective analysis of FP cytotoxicity and aggregation, facilitating the selection of suitable FPs.

Conclusions:

• Engineered FPs with reduced aggregation offer a non-cytotoxic alternative for cellular labeling.
• The new FP variants provide versatile tools for multicolor imaging and flow cytometry in various cell types.
• These advancements enhance the utility of FPs in biomedical research by overcoming limitations of cytotoxicity and aggregation.