Improving the Accumulation and Solubility of Human Interferon-γ (hIFN-γ) in Escherichia coli: A Fusion Protein-Based Method and Network Pharmacology Analysis.

Abstract

Using a network pharmacology-based method, some differentially expressed genes (DEGs) were detected in the colon cancer cell line (HCT116) after treatment with the human interferon-γ (hIFN-γ). Moreover, several pathways including cell cycle, NOD-like receptor signaling pathway, and P53 signaling pathway were identified, indicating the inhibitory effect of IFN-γ on the growth and proliferation of HCT116 cells. To validate in silico results, the hIFN-γ was first produced in Escherichia coli strain Rosetta and its bioactivity was then analyzed by anticancer assay. The production of hIFN-γ was performed via the optimization of several effective factors and the fusion of hIFN-γ to elastin like-polypeptide (ELP) tag. The highest amount of hIFN-γ (3.87 ± 0.37% of total soluble protein) was obtained at 22 °C with OD₆₀₀ = 0.6 and IPTG = 0.25 after 3-h inoculation. Whereas, the highest level of the hIFN-γ-ELP, about 4.58 ± 0.14% of TSP, was observed after 6-h inoculation. Compared to the hIFN-γ, the amount of hIFN-γ-ELP accumulation in the form of soluble increased significantly by more than 18%, proposing the desirable effect of ELP on the accumulation and solubility of hIFN-γ. Furthermore, the hIFN-γ prohibited the growth and proliferation of the HCT116 cells and the highest level of inhibition of cell proliferation was found at a concentration of 32.00 pg/mL hIFN-γ after 72-h incubation. Anticancer activity of hIFN-γ was also confirmed through the expression analysis of Bax, p53, and Bcl-2, suggesting the cytotoxic role of hIFN-γ toward HCT116 cells via inducing apoptosis process and arresting cell cycle.