Dual function labeling of biomolecules based on DsRed-Monomer

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• The lack of fusion tags with both affinity and visualization capabilities hinders biomolecular research.
• Current methods often require separate affinity and reporter tags or dual-terminal fusions.

Purpose of the Study:

• To develop a site-specific genetic fusion approach using DsRed-Monomer as a single tag with dual affinity and reporter functions.
• To demonstrate the utility of this dual-function tag in protein purification and biomolecular assays.

Main Methods:

• Engineered fusion proteins: bradykinin-DsRed-Monomer and calmodulin-DsRed-Monomer.
• Purification using a copper-immobilized column exploiting DsRed-Monomer's copper-binding property.
• Spectroscopic characterization to assess the impact of fusion on DsRed-Monomer properties.
• Developed a competitive fluorescence immunoassay for bradykinin.
• Detected calmodulin ligand binding using calmodulin-DsRed-Monomer via fluorescence changes.

Main Results:

• Successfully prepared and purified bradykinin-DsRed-Monomer and calmodulin-DsRed-Monomer fusion proteins.
• Fusion did not alter the spectroscopic properties of DsRed-Monomer.
• Demonstrated the application of the fusion protein in a bradykinin immunoassay.
• Showcased the detection of chlorpromazine binding to calmodulin through DsRed-Monomer fluorescence changes.

Conclusions:

• DsRed-Monomer serves as an effective dual-function tag, offering both affinity and reporter capabilities.
• This approach simplifies biomolecular research by reducing the need for multiple tags.
• The DsRed-Monomer dual-function tag shows significant potential for applications in proteomics and broader biomolecular research.