[Establishment and functional characterization of an efficient cell-free expression system of bovine...

This study aimed to optimize the pET-23b plasmid structure to enhance the bovine interferon-gamma (IFN-γ) yield in a cell-free expression system. Initially, the gene stability of bovine IFN-γ was improved via bioinformatic mutation, The rop fragment, bom fragment and f1 ori fragment in the original plasmid origin-pET-23b-IFN-γ carrying the optimized bovine IFN-γ gene were deleted by overlapping PCR to construct a high copy number m-pET-23b-IFN-γ plasmid. The m-pET-23b-IFN-γ plasmid was subsequently expressed in Escherichia coli-based cell-free expression system. Western blotting confirmed satisfactory reactivity of the expressed bovine IFN-γ, with expression levels exceeding those from the original pET-23b system. Following centrifugation, the supernatant from the cell-free expression system was collected, using magnetic agarose beads for His-tag protein purification kit to purify the His-tagged protein in the supernatant. The purified recombinant bovine IFN-γ (rIFN-γ) was then evaluated for cell safety, biological activity against Mycobacterium bovis, and transcription levels of interferon-stimulated genes (ISGs) in cells. The results showed that bovine IFN-γ was almost non-toxic to cells at the concentration below 100 μg/mL, had anti-M. bovis activity, and increased the transcription levels of ISGs, which indicated that rIFN-γ had good biological activity. We successfully established an efficient cell-free expression system for bovine IFN-γ and offered a safe and productive pathway for bovine IFN-γ generation, relevant for the diagnosis and treatment of bovine tuberculosis, thus indicating broad application prospects.