Rapid protein-folding assay using green fluorescent protein

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Green fluorescent protein (GFP) chromophore formation requires proper protein folding.
• Protein aggregation is a common challenge during recombinant protein expression in Escherichia coli.
• Assessing protein folding and solubility often necessitates specific functional assays.

Purpose of the Study:

• To develop a "folding reporter" vector for monitoring protein folding.
• To evolve proteins prone to aggregation into soluble and functional variants.
• To establish a directed evolution strategy for improving protein folding and expression.

Main Methods:

• Constructed an N-terminal fusion of test proteins with GFP.
• Utilized fluorescence in Escherichia coli as an indicator of upstream protein folding.
• Applied directed evolution to improve protein folding and solubility.

Main Results:

• Demonstrated a correlation between E. coli cell fluorescence and upstream protein domain folding.
• Successfully evolved aggregation-prone proteins into robustly folding, soluble, and functional forms.
• Validated the folding reporter system across a panel of 20 diverse proteins.

Conclusions:

• The GFP-based folding reporter system effectively indicates protein folding status.
• Directed evolution using this reporter can overcome protein aggregation issues in E. coli.
• This approach offers a simplified method for enhancing protein folding and expression.