Using green and red fluorescent proteins to teach protein expression, purification, and crystallization

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Traditional protein science laboratory curricula often lack engaging, real-time visualization tools.
• Understanding protein expression, purification, and modification is crucial for modern molecular biology.

Purpose of the Study:

• To develop an innovative laboratory curriculum for protein science education.
• To utilize green and red fluorescent proteins (GFP and RFP) for visualizing key protein manipulation techniques.
• To enhance student comprehension and engagement in modern protein science.

Main Methods:

• Amplification and cloning of EGFP and mDsRed coding sequences into prokaryotic expression vectors (pMAL and pT7His).
• Expression of fluorescent proteins in Rosetta (DE3) pLysS using IPTG or autoinduction.
• Purification via multiple chromatography techniques (affinity, ion-exchange, size exclusion, hydrophobic interaction).
• TEV protease cleavage of MBP-EGFP fusion protein and crystallization of mDsRed using hanging drop vapor diffusion.

Main Results:

• Successful cloning, expression, and purification of EGFP and mDsRed fusion proteins.
• Demonstration of various protein purification techniques and protease cleavage.
• Visualization of protein crystallization process in real-time.
• Enhanced clarity and engagement in teaching protein science concepts.

Conclusions:

• The developed curriculum effectively uses fluorescent proteins to illustrate complex protein science experiments.
• Real-time visualization significantly improves student understanding and interest in protein manipulation techniques.
• This approach provides a vivid and accessible platform for learning modern protein science methodologies.