Development of a Method for Producing Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor Using Fusion Protein Technology

• Published in: Current issues in molecular biology Vol. 47; no. 9; p. 681
• Main Authors: Volosnikova, Ekaterina A., Esina, Tatiana I., Volkova, Natalia V., Belenkaya, Svetlana V., Gogina, Yana S., Shimina, Galina G., Vyazovaya, Elena A., Gamaley, Svetlana G., Danilenko, Elena D., Shcherbakov, Dmitriy N.
• Format: Journal Article
• Language: English
• Published: 25.08.2025
• ISSN: 1467-3045; 1467-3045
• DOI: 10.3390/cimb47090681

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifunctional cytokine with therapeutic applications in oncology and neurodegenerative diseases. However, its clinical use is limited by the high cost of eukaryotic production systems. Here, we developed a cost-effective Escherichia coli-based platform for high-yield production of biologically active recombinant human GM-CSF (rhGM-CSF) using SUMO fusion technology. The engineered pET-SUMO-GM plasmid enabled expression of a 33 kDa fusion protein, accounting for 23–25% of total cellular protein, though it primarily accumulated in inclusion bodies. A multi-step purification strategy—including nickel affinity chromatography, Ulp protease cleavage, and hydrophobic chromatography—yielded >99.5% pure rhGM-CSF. In vitro functional assays demonstrated equivalent activity to the WHO international standard (ED50: 0.045 vs. 0.043 ng/mL in TF-1 cell proliferation). In vivo, the preparation significantly restored neutrophil counts (3.4-fold increase, p ≤ 0.05) in a murine cyclophosphamide-induced myelosuppression model. Our results establish a scalable, prokaryotic-based method to produce functional rhGM-CSF, overcoming solubility and folding challenges while maintaining therapeutic efficacy. This approach could facilitate broader clinical and research applications of GM-CSF, particularly in resource-limited settings.