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

TL;DR

This study tests CusF as a solubility tag for proteins but finds it ineffective for two test proteins and inconsistent in binding to purification resins.

Contribution

The study provides new empirical evidence that CusF does not consistently improve solubility or bind Cu-based resins for all proteins.

Findings

CusF fusion did not improve solubility of Efa PAP or mCherry in E. coli.

CusF–mCherry failed to bind Cu2+-charged IMAC resin, with Ni2+ binding due to a His tag.

Results highlight the need to test multiple solubility tags for recombinant protein production.

Abstract

The metal-binding periplasmic protein CusF has been proposed as a bifunctional tag that enhances the solubility of recombinant proteins and enables purification using Cu affinity chromatography. However, evidence for its performance remains limited to a few model proteins. Here, we evaluated CusF as a solubility tag for two heterologous proteins: a putative poly(A)-polymerase from Enterococcus faecalis (Efa PAP) and the red fluorescent protein mCherry. The proteins were fused to CusF, expressed in E. coli BL21 (DE3) pLysS and Rosetta 2 (DE3) strains, and assessed for solubility and IMAC binding. Native Efa PAP was completely insoluble under all tested conditions, and fusion to CusF did not improve its solubility. Similarly, CusF–mCherry accumulated predominantly in the insoluble fraction, with only trace amounts detectable in soluble lysates. Soluble CusF–mCherry did not bind Cu2+-charged IMAC resin, while moderate binding to Ni2+-charged resin was attributable to the vector-encoded His tag rather than CusF. These results indicate that CusF does not universally enhance protein solubility and may not consistently bind Cu-based IMAC resin. Our findings expand empirical knowledge of solubility tag performance and emphasize the necessity of testing multiple tags to identify optimal strategies for recombinant protein production.

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