Soluble Expression of Human Granulocyte Colony Stimulating Factor (hG-CSF) in Escherichia coli Expression System
Abstract
Human granulocyte colony stimulating factor (hG-CSF) is a hematological growth factor that plays a crucial role in neutrophil production and differentiation. Some foreign biomolecules, especially of human origin, such as hG-CSF, sometimes aggregate because of different factors during expression and create inclusion bodies in Escherichia coli (E. coli). Refolding process is commonly used to recover these very valuable molecules, but still significant amounts of protein remain unusable. Refolding procedures are frequently costly, time-consuming, and not fully efficient. Therefore, the use of molecular chaperones to improve soluble expression of proteins was evaluated in the study. In this context, hG-CSF was co-expressed with five chaperone plasmid systems (pGro7, pG-KJE8, pG-Tf2, pKJE7, pTf16) to ensure the expression of hG-CSF in soluble form. Among these, the pKJE7 plasmid was found to be the most effective in obtaining hG-CSF in soluble form, yielding 92% purity after Ni-NTA affinity chromatography purification. The total yield of hG-CSF obtained was 1.6 mg per 1 L bacterial culture. The biological activity of the soluble hG-CSF was evaluated in human umbilical vein endothelial cells (HUVECs). A 24-hour interaction of hG-CSF with HUVECs resulted in a significant increase in cell viability at all applied doses, demonstrating its bioactivity. As a result, hG-CSF, which previously aggregated as an inclusion body in the E. coli expression system, was correctly folded by co-expression with chaperone proteins were obtained as more efficient and purer.
Keywords
Granulocyte colony stimulating factor, Recombinant proteins, Molecular chaperones, E. coli
Supporting Institution
Tokat Gaziosmanpaşa University
Project Number
2020/67
Thanks
The study was funded by Tokat Gaziosmanpaşa University, Foundation of Scientific Research Projects (Project number: 2020/67).
References
- Babaeipour, V., Khanchezar, S., Mofid, M. R., and Abbas, M. P. H. (2015). Efficient process development of recombinant human granulocyte colony-stimulating factor (rh-GCSF) production in Escherichia coli. Iranian biomedical journal, 19(2), 102.
- Babaeipour, V., Khanchezar, S., Mofid, M. R., and Pesaran Hagi Abbas, M. (2015). Efficient process development of recombinant human granulocyte colony-stimulating factor (rh-GCSF) production in Escherichia coli. Iran Biomed J, 19(2), 102-110.
- Basu, S., Dunn, A., and Ward, A. (2002). G-CSF: function and modes of action (Review). Int J Mol Med, 10(1), 3-10.
- Bozkurt, Y., Bilgin, S., Erden, S., Turan, İ. F., and Gökçe, İ. (2019). Recombinant human G-CSF production as a protein based drug candidate for hematology and oncology. International Journal of Chemistry and Technology, 3(2), 92-100.
- Cerchione, C., Nappi, D., and Martinelli, G. (2021). Pegfilgrastim for primary prophylaxis of febrile neutropenia in multiple myeloma. Support Care Cancer.
- Coward, J. I., Nathavitharana, R., and Popat, S. (2012). True hypoglycaemia secondary to treatment with granulocyte colony stimulating factor (G-CSF) in a diabetic patient with non-small cell lung cancer. Lung Cancer, 75(1), 133-135.
- Do, B. H., Kang, H. J., Song, J. A., Nguyen, M. T., Park, S., Yoo, J., et al. (2017). Granulocyte colony-stimulating factor (GCSF) fused with Fc Domain produced from E. coli is less effective than Polyethylene Glycol-conjugated GCSF. Sci Rep, 7(1), 6480.
- Eczacioglu, N., Ulusu, Y., Gokce, İ., and Lakey, J. H. (2022). Investigation of mutations (L41F, F17M, N57E, Y99F_Y134W) effects on the TolAIII-UnaG fluorescence protein's unconjugated bilirubin (UC-BR) binding ability and thermal stability properties. Preparative Biochemistry & Biotechnology, 52(4), 365-374.
- Francis, D. M., and Page, R. (2010). Strategies to optimize protein expression in E. coli. Curr Protoc Protein Sci, Chapter 5(1), 5.24.21-25.24.29.
- İncir, İ., and Kaplan, Ö. (2024). Escherichia coli as a versatile cell factory: Advances and challenges in recombinant protein production. Protein Expr Purif, 219, 106463.
