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 Cu(2+)-charged IMAC resin, while moderate binding to Ni(2+)-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.