Versatile format of minichaperone-based protein fusion system

Area of Science:

• Biotechnology
• Protein Engineering
• Structural Biology

Background:

• Hydrophobic recombinant proteins frequently aggregate into inclusion bodies, hindering soluble production and refolding.
• A fusion system utilizing the thermophilic minichaperone, GroEL apical domain (GrAD), has been developed to facilitate soluble protein production.
• Protein engineering strategies are crucial for overcoming challenges in recombinant protein expression and purification.

Purpose of the Study:

• To engineer and evaluate permutated variants of the GroEL apical domain (GrAD) for enhanced fusion protein production.
• To assess the efficacy of the GrAD fusion system in producing soluble and refoldable forms of hydrophobic proteins, specifically the hepatitis C virus E2 protein N-terminal fragment.
• To demonstrate the flexibility of the engineered GrAD system for optimizing interactions with various target protein moieties.

Main Methods:

• Gene engineering was employed to create permutated GrAD variants by relinking N- and C-termini and introducing new termini on the protein surface.
• Fusion constructs were designed, combining GrAD and its permutated forms with the insoluble N-terminal fragment of the hepatitis C virus E2 protein.
• Expressed fusion proteins were analyzed for solubility and refolding efficiency after denaturation and renaturation processes.

Main Results:

• Engineered fusion proteins, including those with permutated GrAD variants and the hepatitis C virus E2 protein fragment, initially formed inclusion bodies upon expression.
• Despite initial aggregation, all tested fusion proteins demonstrated complete renaturation into stable, soluble forms after denaturation and subsequent refolding.
• The study successfully generated and analyzed two distinct permutated GrAD forms, highlighting the system's adaptability.

Conclusions:

• The developed GrAD fusion system, including engineered permutated variants, effectively facilitates the production of soluble and refoldable hydrophobic proteins.
• The strategy of creating permutated minichaperone variants offers significant flexibility in optimizing the carrier-target protein interaction for diverse applications.
• This versatile system provides a valuable platform for addressing the common bottleneck of insoluble protein production in biotechnology and research.