Split-enzyme fragment as a single affinity tag that enables protein expression, purification, and functional assays

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Science

Background:

• Recombinant protein production is vital across biological sciences.
• Current methods often require multiple molecular tags for expression, purification, and characterization.
• A need exists for a single, versatile tag that simplifies the entire protein production process.

Purpose of the Study:

• To develop and validate a single molecular tag system for comprehensive recombinant protein production.
• To adapt a split-luciferase system for real-time expression monitoring, affinity purification, and downstream characterization.
• To demonstrate the utility of a single tag in overcoming limitations of multi-tag approaches.

Main Methods:

• Integration of a split-luciferase system (NanoBiT®) for protein tagging.
• Development of real-time protein expression monitoring techniques.
• Construction of a novel affinity chromatography system utilizing the split-luciferase tag.
• Adaptation of Western blot, ELISA, and cell-based bioassays for protein characterization.

Main Results:

• A simple method for real-time monitoring of protein expression was established.
• A novel affinity purification system based on the split-luciferase tag was successfully constructed.
• The split-luciferase tag enabled effective characterization of expressed proteins using various assays.
• Demonstrated that a single tag can fulfill all essential steps in protein production.

Conclusions:

• A single split-luciferase tag can comprehensively support recombinant protein production from expression to characterization.
• This integrated system offers a streamlined and efficient alternative to multi-tag strategies.
• The developed method enhances the workflow for producing and analyzing recombinant proteins in research and industry.