A standardized set of pNX vectors for enhanced soluble expression of recombinant proteins in E. coli using small fusion tags

Background The production of recombinant proteins in Escherichia coli ( E. coli ) is often hampered by the formation of inclusion bodies. While fusion tags can enhance solubility, existing systems are hampered by a lack of standardization, with tags scattered across disparate plasmid backbones and inconsistent cloning sites, complicating high-throughput screening. Results To address this, we constructed a standardized series of expression vectors, termed pNX, by incorporating nine small fusion tags (SUMO, LD, ACP, BCCP, GB1, Fh8, SmbP, TolA, and TrxA) into a uniform pET-28b backbone. Each pNX vector features an identical configuration: a T7 promoter, an N-terminal fusion tag, a 6×His tag, a linker, a TEV protease cleavage site, a multiple cloning site (MCS), and a C-terminal 6×His tag. We evaluated this system using four model proteins (EcFabG, eGFP, XccXanA2, and XccXanL). Our results showed that specific tags significantly improved both the expression level and solubility of the target proteins without compromising their biological activity. Notably, the lipoyl domain (LD) was identified for the first time as an effective solubility enhancer. The standardized MCS enabled rapid, parallel cloning, facilitating the efficient screening of optimal fusion partners. Conclusions The pNX vector series provides a versatile and high-throughput platform for enhancing the soluble expression of challenging recombinant proteins in E. coli , streamlining the empirical identification of ideal fusion tags.