Soluble Expression of a Membrane-Bound Linoleic Acid Isomerase Using a Fusion Tag Strategy

The membrane-bound linoleic acid isomerase from B. breve CCFM683 (BBI) is a key biocatalyst for the conversion of linoleic acid (LA) to conjugated linoleic acid ( c 9, t 11-CLA). However, its intrinsic membrane-associated nature severely limits the efficiency of heterologous expression. To address this, we employed a fusion tag strategy to enhance solubility and improve protein expression levels. We introduced the solubilizing tag NusA and a FLAG tag for detection, systematically evaluating their effects on BBI expression and function. Protein solubility and CLA bioconversion activity were assessed through Western blot analysis of recombinant proteins and GC-MS quantification of products. The results demonstrated that NusA increased BBI expression, raising the soluble fraction from 9.52% to 66.49%, but significantly reduced enzymatic activity. To reconcile the trade-off between solubility and activity, we incorporated a flexible linker peptide between BBI and NusA. This modification enhanced total BBI expression by 3.49-fold, increased the soluble ratio to 95.06%, and restored CLA bioconversion activity to 97.23%, representing a 3.82-fold improvement compared to the construct without the linker. Molecular docking analysis suggested that the underlying mechanism may involve steric hindrance imposed by NusA on BBI, which was alleviated by the physical separation afforded by the linker. This study highlights the regulatory role of domain conformational flexibility in transmembrane protein function and offers an effective strategy for the soluble expression and rational design of complex membrane proteins.