OPTIMIZATION OF REFOLDING CONDITIONS FOR RECOMBINANT OMP19 PROTEIN FROM BRUCELLA SPP.

The recombinant Omp19 protein of Brucella spp. is considered as a promising antigen for the development of diagnostic test systems and immunobiological preparations. However, obtaining its soluble form remains a significant challenge. The aim of this study was to optimize the dialysis conditions for the recombinant Omp19 protein following purification by ion-exchange chromatography.
Dialysis was performed sequentially in several buffer systems with varying compositions of salts and stabilizing components. Dialysis efficiency and protein solubility were assessed using the SDS-PAGE method, with analysis of the supernatant and pellet fractions under various storage temperature conditions. The results showed that when buffers 1-4 were used, the recombinant protein predominantly converted to an insoluble form and was detected in the pellet. To increase the protein’s solubility, the concentration of L-arginine in the dialysis buffer was optimized. It was found that using a buffer containing 880 mM Larginine ensured that the protein remained in the soluble fraction and prevented the formation of a visible pellet.
Based on these results, we recommend using 880 mM L-arginine to stabilize the recombinant Omp19 protein during purification and storage, which is of practical importance for the further use of this protein in diagnostics and biotechnology research.

1. Pappas G., Papadimitriou P., Akritidis N., Christou L., Tsianos E. V. The new global map of human brucellosis // Lancet Infectious Diseases. – 2006. – Vol. 6, No. 2. – P. 91-99.

2. Tibor A., Weynants V., Denoel P., Schmitt D., van Broeck J., Letesson J. J. Outer membrane proteins Omp10, Omp16 and Omp19 of Brucella spp. are lipoproteins // Infection and Immunity. – 1999. – Vol. 67, No. 9. – P. 4960-4962.

3. Baneyx F., Mujacic M. Recombinant protein folding and misfolding in Escherichia coli // Nature Biotechnology. – 2004. – Vol. 22, No. 11. – P. 1399-1408.

4. Arakawa T., Tsumoto K. The effects of arginine on protein folding and aggregation // Biochemical and Biophysical Research Communications. – 2003. – Vol. 304, No. 1. – P. 148-152.

5. Bulashev A.K., Akibekov O.S., Syzdykova A.A. et al. Use of recombinant Brucella outer membrane proteins for serological diagnosis of brucellosis // Veterinary World. – 2020. – Vol. 13, No. 7. – P. 1439-1447.

6. Bulashev AK, Akibekov OS, Syzdykova AA, et al. Use of recombinant Brucella proteins for the serodiagnosis of bovine brucellosis. Bulletin of S. Seifullin Kazakh Agrotechnical University. 2020;(2):45– 52. [In Russian].

7. Wingfield P.T. Protein purification and characterization. In: Current Protocols in Protein Science. Hoboken: Wiley; 2024.

8. Wingfield P. T. Protein purification and refolding // Current Protocols in Protein Science. – Hoboken: Wiley, 2024.

9. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 // Nature. – 1970. – Vol. 227. – P. 680-685.

10. Kurien B.T., Scofield R.H. SDS-PAGE. In: Protein Electrophoresis: Methods and Protocols. Methods in Molecular Biology. New York: Humana Press; 2023. P. 45-58.

11. Neuhoff V., Arold N., Taube D., Ehrhardt W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250 // Electrophoresis. – 1988. – Vol. 9, No. 6. – P. 255-262.

12. Rahban M., Ahmad F., Piatyszek M.A., Haertlé T., Saso L., Saboury A.A. Stabilization challenges and aggregation in protein-based therapeutics in the pharmaceutical industry // RSC Advances. – 2023. – Vol. 13. – P. 35947-35963. DOI: 10.1039/D3RA06476J.

13. Lee J.M., Hammarén H.M., Savitski M.M., Baek S.H. Control of protein stability by posttranslational modifications // Nature Communications. – 2023. – Vol. 14. – Art. 201. DOI: 10.1038/s41467-023-35795-8.

14. Tibor A., Decelle B., Letesson J.J. Outer membrane proteins Omp10, Omp16, and Omp19 of Brucella spp. are lipoproteins // Infection and Immunity. 1999. Vol. 67, No. 9. P. 4960-4962. DOI: 10.1128/IAI.67.9.4960-4962.1999.

15. Golchin M., Etemadi J., Moazeni Jula G. et al. Serodiagnosis of human brucellosis by an indirect ELISA test using recombinant outer membrane protein 19 kDa (rOMP19) as an antigen // BMC Biotechnology. 2023. Vol. 23. Art. 46. DOI: 10.1186/s12896-023-00817-2.

16. Zhang Y., Wang X., Li Y. et al. Optimizing ovine brucellosis serodiagnosis: evaluation of recombinant Brucella antigens and multi-antigen combinations for iELISA // Frontiers in Veterinary Science. 2026. Vol. 13. Art. 1775573.

17. Ren S. Effects of arginine in therapeutic protein formulations: a decade review and perspectives // Antibody Therapeutics. 2023. Vol. 6, No. 4. P. 265–276. DOI: 10.1093/abt/tbad022.

18. Gupta M.N., Uversky V.N. Biological importance of arginine: a comprehensive review of the roles in structure, disorder, and functionality of peptides and proteins // International Journal of Biological Macromolecules. 2024. Vol. 257. Art. 128646. DOI: 10.1016/j.ijbiomac.2023.128646.