A standardized set of pNX vectors for enhanced soluble expression of recombinant proteins in E. coli using small fusion tags
Year: 2025
Type: article
Abstract: 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 parallel 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 synthetic linker, a TEV protease cleavage site, and a multiple cloning site (MCS) flanked by dual 6×His tags. 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, to our knowledge 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 efficient platform for enhancing the soluble expression of challenging recombinant proteins in E. coli, streamlining the empirical identification of ideal fusion tags.
Source: Microbial Cell Factories
Authors: Li-Zhen Luo, Wen-Bin Zhang, Zhe Hu, Ling-Hua Zhang, Jin-Cheng Ma, Xue-Bin Jiang
Institutions: South China Agricultural University, Guangzhou University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,590
Award: G4688188029
OpenAlex ID: W4417488560