Production and purification of modified recombinant SMN1 protein with potential use in spinal muscular atrophy treatment and ability to cross the cell membrane

Aim: Producing Modified Recombinant SMN1 Protein and Ensuring Its Passage Through the Cell Membrane. However, this study does not include preclinical or clinical trials; it will only investigate the production of the protein and its potential to cross the cell membrane in a cell culture environment. Material and Method: The study primarily involved the production of a recombinant protein composed of PEP1 (signal peptide) + SMN1 protein + 6x histidine. The vector was prepared through either molecular cloning or gene synthesis methods. After the vector was confirmed by DNA sequencing analysis, it was transferred into E. coli BL21 strain to facilitate the expression of the target protein in bacteria. Protein production was induced with IPTG, and the protein was subsequently purified using a commercial metal affinity chromatography kit with the 6xHis tag. The amount of the purified protein was determined using the Bradford assay, and its purity was assessed by SDS-PAGE analysis. Human neuronal cell lines (SHSY5Y) were used in the study. The cells were cultured in a medium suitable for neuronal cells, consisting of DMEM and Ham's F12 with 10% fetal bovine serum. The entry of the protein into the cells, added in varying amounts, was examined at different time intervals using Western blotting with a 6xHis specific antibody. Results: Based on the analysis results, it was observed that the potential of the PEP1-SMN1 recombinant protein to pass through the cell membrane was successfully confirmed. The induction process determined that the optimal conditions were incubation with 0.5 mM IPTG at 30°C for 4 hours. Western blot analyses showed a band close to the theoretical weight of the protein, and protein purity was enhanced using urea buffers. In cell culture experiments, positive results were obtained, supporting cell adhesion and membrane translocation of the protein. Conclusion: n this study, it was demonstrated that the PEP1-SMN1 recombinant protein was successfully produced and purified as a potential therapeutic agent capable of crossing the cell membrane. The findings indicate that the optimized methods for protein induction and purity were effective, supporting the in vitro activity of the protein. These results provide significant contributions that could form the basis for future preclinical studies on Spinal Muscular Atrophy treatment. Key words: SMN1, IPTG, BL21, SMA, DMEM, FBS, PEP1