Engineering an EGFR-binding Gp2 domain for increased hydrophilicity.

Area of Science:

• Protein Engineering
• Biochemistry
• Molecular Imaging

Background:

• The Gp2 domain is a 45 amino-acid scaffold utilized for high-affinity binding in molecular imaging and biological antagonism.
• Increased hydrophilicity is hypothesized to improve Gp2's physiological distribution and physicochemical robustness.

Purpose of the Study:

• To enhance the hydrophilicity of the Gp2 domain through targeted mutations.
• To evaluate the impact of these mutations on Gp2's expression, binding affinity, specificity, solubility, and thermal stability.

Main Methods:

• Identification of exposed hydrophobic sites on the Gp2 domain for hydrophilic mutations.
• Experimental evaluation of single and multiple point mutants.
• Assessment of hydrophilicity using reverse-phase high-performance liquid chromatography.
• Analysis of binding affinity (Kd), recombinant yield, and thermal stability (Tm).

Main Results:

• Six single mutations were identified that did not significantly impair expression, binding affinity, specificity, or thermal stability.
• Multiple mutations improved hydrophilicity, with one variant (five mutations) showing enhanced solubility, reasonable binding affinity (Kd = 53-63 nM), and good yield (1.3 mg/L).
• Another variant (three mutations) demonstrated increased solubility and nominally improved binding affinity (Kd = 13-28 nM) but had reduced yield.
• A single mutation (T21N) increased thermal denaturation midpoint by ≥7°C.

Conclusions:

• Hydrophobic-to-hydrophilic mutations in the Gp2 domain offer a strategy to improve physicochemical properties.
• Engineered Gp2 variants exhibit enhanced solubility and hydrophilicity, beneficial for molecular imaging probes.
• These modified Gp2 scaffolds provide a foundation for developing new ligands targeting additional biological targets.