Insights into the Performance of CusF as a Solubility Tag for Recombinant Protein Expression

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• The CusF protein is a metal-binding periplasmic protein.
• It has been proposed as a bifunctional tag for enhancing recombinant protein solubility and enabling copper affinity chromatography.
• Evidence for CusF's performance as a tag is limited.

Purpose of the Study:

• To evaluate CusF as a solubility tag for two heterologous proteins: Efa PAP and mCherry.
• To assess CusF's ability to enhance protein solubility and facilitate purification via IMAC.
• To expand empirical knowledge on solubility tag performance.

Main Methods:

• Fusion of CusF to Efa PAP and mCherry.
• Expression of recombinant proteins in E. coli strains (BL21 (DE3) pLysS and Rosetta 2 (DE3)).
• Assessment of protein solubility and IMAC binding to Cu2+ and Ni2+ resins.

Main Results:

• CusF fusion did not improve the solubility of Efa PAP or mCherry.
• CusF-mCherry predominantly accumulated in the insoluble fraction.
• Soluble CusF-mCherry did not bind to Cu2+-charged IMAC resin; binding to Ni2+-charged resin was due to the His tag.

Conclusions:

• CusF does not universally enhance protein solubility.
• CusF may not consistently bind to copper-based IMAC resin.
• Testing multiple solubility tags is crucial for optimizing recombinant protein production.