A modular set of prokaryotic and eukaryotic expression vectors

Area of Science:

• Molecular Biology
• Protein Expression and Purification

Background:

• Efficient purification of recombinant fusion proteins is crucial for biochemical and structural studies.
• Existing expression vectors often face challenges with low expression rates and incomplete tag removal.

Purpose of the Study:

• To develop a modular series of versatile expression vectors for improved affinity purification of recombinant fusion proteins.
• To enhance protein purity, facilitate tag cleavage, and enable expression in multiple host systems.

Main Methods:

• Utilized serial affinity tags (hexahistidine-GST) for enhanced purification.
• Employed hexahistidine-tagged tobacco etch virus (TEV) protease for efficient tag cleavage.
• Designed PCR cloning for specific N-terminal amino acid residues post-cleavage.
• Enabled expression in Escherichia coli and Saccharomyces cerevisiae.

Main Results:

• Achieved extremely pure protein even with very low expression rates.
• Demonstrated highly efficient proteolytic cleavage of affinity tags under various conditions.
• Confirmed purification and biological activity of yeast GAL regulon components using the developed vectors.

Conclusions:

• The described expression vectors offer a versatile and efficient system for recombinant protein purification.
• The modular design and features facilitate high-purity protein production and characterization.
• These vectors are valuable tools for researchers working with low-expression proteins or requiring stringent purity standards.