Optimization of Recombinant Human Interferon-Gamma Expression and Purification in E. coli, and Developing an Immunization Protocol for the Production of Optimal Humoral Response

Background: Recombinant human interferon gamma (rhIFN-γ) and monoclonal antibodies are promising biomolecules for improving clinical medicines worldwide due to their biomedical applications. However, most African countries lack established production protocols. Although Freund’s Complete Adjuvant (FCA) has been widely used to enhance antibody production, it has been reported to cause adverse effects. This study aimed to optimize the expression and purification of rhIFNγ and develop an immunization protocol for producing optimal humoral responses using alternative adjuvants such as RIBI Immunochem (RIBI) adjuvant and Freund’s Incomplete Adjuvant (FIA), with FCA as the standard. Methodology: The human interferon gamma (NM000619.3) sequence was retrieved from NCBI and sent to GenScript for codon optimization and construct synthesis. The constructs were transformed into E. coli BL-21, and protein expression was induced with IPTG. The expression and purification conditions were optimized. To evaluate immunization regimens and alternative adjuvants to FCA, three groups of female BALB/c mice were immunized with varying concentrations of rhIFNγ (25, 50, and 100 μg) mixed with an equal volume of RIBI adjuvant, FIA, and FCA. An Enzyme-Linked Immunosorbent Assay was used to measure antibody levels from each group. Results: The study identified 0.6 mM of IPTG at 37°C and 250 rpm for 4 hours as the optimal conditions for rhIFNγ expression. Increasing imidazole concentration in wash and elution buffers enhanced rhIFNγ purity but reduced protein yield. Administration of 100 μg of rhIFNγ with RIBI adjuvant elicited a sufficient immune response, while higher concentrations of FCA and FIA increased side effects, indicating the need for lower adjuvant doses. Conclusion: Although FCA has been reported to produce high antibody titers with notable side effects, this study found no significant difference in antibody levels between mice immunized with 100 μg rhIFNγ mixed with FCA and the group immunized with 100 μg rhIFNγ plus RIBI adjuvant. Keywords: recombinant human interferon-gamma, RIBI immunochem adjuvant, Freund’s incomplete adjuvant, humoral response