Spinal müsküler atrofi tedavisinde kullanılma potansiyeline sahip ve hücre membranından geçebilen modifiye rekombinant SMN1 proteininin üretilmesi ve saflaştırılması
Küçük Resim Yok
Tarih
2024
Yazarlar
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Cilt Başlığı
Yayıncı
İnönü Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Amaç: PEP1-SMN1 proteinini modifiye rekombinant şekilde üretip hücre membranından geçişini sağlamak. Fakat bu çalışma kapsamında preklinik ya da klinik çalışma bulunmamaktadır sadece proteinin üretimi ve hücre kültürü ortamında hücre membranından geçme potansiyeli araştırılmıştır. Materyal Metod: Çalışmada temel olarak PEP1 (sinyal peptidi) + SMN1 proteini + 6x histidin yapısında rekombinant protein üretimi yapıldı. Vektörün hazırlanması gen sentezi yollarından birisi ile yapıldı. Oluşturulan vektör DNA sekans analizi ile doğrulandıktan sonra E. coli BL21 suşuna aktarılırak ilgili proteinin bakteride ifade edilmesi sağlandı. Protein üretimi IPTG ile indüklendi ve sonrasında 6XHis işareti kullanılarak ticari bir metal afinite kromotografisi kiti ile saflaştırıldı. Saflaştırılan proteinin miktar tayini Bradford testi ile ve saflık derecesi SDS-PAGE analizi incelendi. Çalışmada İnsan nöron kökenli hücre hatları kullanıldı. (SHSY5Y). Besiyeri olarak nöron hücreleri için uygun olan DMEM ve Ham's F12 ortamı ve %10 ek fetal bovine serum kullanıldı. Farklı miktarlarda hücre kültürüne eklenen proteinin farklı zaman aralıklarında hücre içine girişi 6XHis spesifik antikor kullanılarak Western blotlama analizleri ile gösterildi. Bulgular: Yapılan analizler sonucunda, PEP1-SMN1 rekombinant proteininin hücre membranından geçme potansiyelinin başarılı bir şekilde doğrulandığı gözlemlendi. İndüksiyon sürecinde 0.5 mM IPTG ve 30°C'de 4 saatlik inkübasyonun en uygun şartlar olduğu belirlendi. Western blot analizleri, proteinin teorik ağırlığına yakın bir bant gösterdi. Hücre kültür deneylerinde, proteinin hücre yapışmasını ve membran geçişini destekleyen olumlu sonuçlar elde edildi. Sonuçlar: Bu çalışmada, PEP1-SMN1 rekombinant proteininin hücre membranından geçebilen potansiyel bir terapötik ajan olarak başarılı bir şekilde üretildiği ve saflaştırıldığı gösterilmiştir. Elde edilen sonuçlar, protein indüksiyonu ve saflığı için optimize edilen yöntemlerin etkili olduğunu göstermektedir. Bu bulgular, Spinal Musküler Atrofi tedavisi için gelecekteki preklinik araştırmaların temelini oluşturabilecek nitelikte önemli katkılar sunmaktadır. Anahtar kelimeler: SMN1, IPTG, BL21, SMA, DMEM, FBS, PEP1
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
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
Açıklama
Anahtar Kelimeler
Tıbbi Biyoloji, Medical Biology
