Engineering and characterization of a superfolder green fluorescent protein

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Green fluorescent protein (GFP) variants are widely used in molecular biology.
• Misfolding of GFP fusion proteins can complicate the analysis of protein expression and folding.
• Existing enhanced GFP variants still exhibit limitations when fused to challenging polypeptides.

Purpose of the Study:

• To engineer a novel GFP variant with superior folding capabilities when fused to other proteins.
• To assess the performance of this new variant, termed 'superfolder' GFP, in comparison to existing folding-enhanced GFPs.
• To elucidate the structural basis for the improved folding properties of superfolder GFP.

Main Methods:

• Engineering of a new GFP variant ('superfolder' GFP) with enhanced folding properties.
• Comparative analysis of superfolder GFP and 'folding reporter' GFP when fused to various proteins.
• Assessment of fluorescence proportionality to protein expression and folding yield in Escherichia coli.
• X-ray crystallographic structural analysis of superfolder GFP.

Main Results:

• Superfolder GFP demonstrates robust folding even when fused to poorly folded polypeptides.
• It exhibits improved tolerance to circular permutation, enhanced resistance to chemical denaturants, and faster folding kinetics compared to folding reporter GFP.
• Fluorescence in Escherichia coli cells expressing superfolder GFP fusions correlated with total protein expression.
• In contrast, folding reporter GFP fusion fluorescence correlated with productive folding yield.

Conclusions:

• Superfolder GFP is a highly robust and reliable reporter for protein expression analysis.
• Its superior folding properties make it advantageous for studying proteins prone to misfolding.
• Structural insights explain the enhanced folding efficiency of superfolder GFP, providing a basis for further protein engineering efforts.