Enhanced protein expression in the baculovirus/insect cell system using engineered SUMO fusions.

Area of Science:

• Biotechnology
• Molecular Biology
• Protein Engineering

Background:

• Recombinant protein expression in insect cells is highly variable.
• Existing fusion tags aid detection and purification but can be processed by native proteases in eukaryotic systems.
• The small ubiquitin-related modifier (SUMO) tag enhances expression in E. coli but is susceptible to cleavage in eukaryotic systems.

Purpose of the Study:

• To develop and evaluate a novel SUMO-derived fusion tag, SUMOstar, for enhanced recombinant protein expression in insect cells.
• To assess the SUMOstar tag's ability to improve expression levels and maintain protein activity.
• To overcome limitations of the standard SUMO tag in eukaryotic expression systems.

Main Methods:

• Development of the SUMOstar tag, resistant to native SUMO proteases.
• Fusion of SUMOstar to various test proteins: mouse UBP43, human tryptase beta II, USP4, USP15, and GFP.
• Expression of SUMOstar-tagged proteins in a baculovirus/insect cell system.
• Comparison of expression levels with native and His(6)-tagged proteins.
• Assessment of protein activity after expression and purification.

Main Results:

• Fusion to SUMOstar significantly enhanced protein expression levels, at least 4-fold higher than native or His(6)-tagged proteins.
• Active SUMOstar-tagged proteins (UBP43, USP4, USP15, GFP) were successfully isolated.
• Tryptase activity was restored after cleavage with a SUMOstar-specific protease, indicating successful tag removal and protein refolding.

Conclusions:

• The SUMOstar tag is an effective tool for enhancing recombinant protein expression in insect cells.
• SUMOstar fusion technology facilitates the production of difficult-to-express proteins and proteins requiring an intact N-terminal residue.
• This system offers a valuable alternative to existing tags, particularly for therapeutic protein production and structural studies.