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dc.contributor.advisorÇelik Akdur, Eda
dc.contributor.authorCilasun, Selen
dc.date.accessioned2023-06-05T11:29:03Z
dc.date.issued2022
dc.date.submitted2022-12-13
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dc.identifier.urihttps://hdl.handle.net/11655/33279
dc.description.abstractAntibodies and antibody fragments are used in the diagnosis, imaging, and target-specific treatments of various diseases such as cancer and viral infections. It also has an important place in combating infectious diseases. Single-chain antibody fragments (scFv), which consist of a flexible peptide fragment and the V portion of a heavy chain linked to the V portion of a light chain, are smaller in size, can be produced more cheaply and easily in bacterial systems, and have a high selectivity against the target antigen, similar to antibodies, and due to their affinity, they have recently become an alternative to full-antibodies. Biopharmaceutical proteins make up nearly half of the global pharmaceutical market. The majority of biopharmaceutical products include monoclonal antibody and antibody fragments. It is known that Escherichia coli, which is widely used in recombinant protein production, reduces production costs, increases productivity and is easier to manipulate compared to other microorganisms. There is a lack of study in literature in order to determine the optimum production site of anti-HER2 scFv containing disulfide bonds, in which the productions in three different compartments of E. coli are coexisting, comparing using different signal peptides and strains, via different pathways specific to these regions. In the thesis study, productions of selective anti-HER2 scFv against human epidermal growth receptor 2 (HER2), which occur in excessive amounts in breast cancer cells, were compared for three different compartments, periplasmic, cytoplasmic and extracellular. The commercial and genetically modified E. coli SHuffle T7 Express strain was used for the cytoplasmic production. Designs including DsbA and MBP signal peptides were used for periplasmic region, while the design with the YebF signal peptide was used for extracellular secretion. It has been shown in the study that the scFvs purified at a ratio of >99% after production are in soluble and active form. The most suitable temperature for production was determined by screening the induction temperature. After optimizing the production process, scFvs were compared in different features. Periplasmic, cytoplasmic, and extracellular samples whose specificities were evaluated were correctly bound to antigen in the in vitro assays. While the production yield of the cytoplasmic sample was 6.2 mg/L, the yield of periplasmic production with spMBP was 5.1 mg/L and the extracellular production efficiency was 2.5 mg/L. In addition, when the specific heat and melting temperatures (Tm) were examined, it is concluded that the cytoplasmic sample is thermally more stable. Overall, this study has been a guide for the process of reducing the production costs of recombinant proteins that play a role in medical diagnosis and treatment. In particular, the production of anti-HER2 scFv in active and soluble form, its cloning in E. coli using complex protein-specific methods and strains, and evaluation of its different aspects will make an important contribution to the literature.tr_TR
dc.language.isoturtr_TR
dc.publisherFen Bilimleri Enstitüsütr_TR
dc.rightsinfo:eu-repo/semantics/openAccesstr_TR
dc.subjectAntikor fragmanıtr_TR
dc.subjectE. coli
dc.subjectSinyal peptit
dc.subjectRekombinant protein
dc.subjectPeriplazmik
dc.subjectEkstraselüler
dc.subjectSitoplazmik
dc.subject.lcshKimya mühendisliğitr_TR
dc.titleAnti-Her2 SCFV Antikor Fragmanının Escherichia coli Bakterisinde Üretim Bölgesinin Optimizasyonutr_TR
dc.typeinfo:eu-repo/semantics/masterThesistr_TR
dc.description.ozetAntikorlar ve antikor fragmanları, kanser ve viral enfeksiyonlar gibi çeşitli hastalıkların tanısında, görüntülemede ve hedefe özgü tedavilerde kullanılmaktadır. Bulaşıcı hastalıklarla mücadelede de önemli bir yere sahiptir. Esnek bir peptit parçası ile bir hafif zincirin V kısmına bağlanmış bir ağır zincirin V kısmından oluşan tek-zincir antikor fragmanları (scFv), daha küçük boyutlarda olması, bakteri sistemlerinde daha ucuz ve kolaylıkla üretilebilmesi ve hedef antijene karşı, antikorlara benzer düzeyde, yüksek seçicilik ve afinite göstermelerinden dolayı, son zamanlarda tam-antikorlar yerine kullanılabilmektedir. Biyofarmasötik proteinler küresel ilaç pazarının yaklaşık yarıya yakınını oluşturmaktadır. Biyofarmasötik ürünlerin çoğunluğunu monoklonal antikor ve antikor fragmanları kapsamaktadır. Rekombinant protein üretiminde yaygın olarak kullanılan Escherichia coli’nin, üretim maliyetlerini düşürdüğü, verimliliği arttırdığı ve diğer mikroorganizmalara kıyasla manipülasyonu daha kolay olduğu bilinmektedir. Literatürde, disülfit bağı içeren anti-HER2 scFv’nin optimum üretim yerinin belirlenmesine yönelik E. coli’nin üç farklı bölmesindeki üretimlerin bir arada olduğu, bu bölgelere özgü farklı yolakları kullanan birbirinden farklı sinyal peptitleri ve suş kullanılarak karşılaştırılmasına ve üç bölme arasında optimum üretim yerinin belirlenmesine yönelik bir çalışma bulunmamaktadır. Yapılan tez çalışmasında, meme kanseri hücrelerinde normalden fazla miktarda ortaya çıkan insan epidermal büyüme reseptörü 2 (HER2)’ye karşı seçici anti-HER2 scFv’nin, genetiği değiştirilmiş ticari E. coli bakterisinin SHuffle T7 Express suşunda sitoplazmik bölgede üretimi, BL21(DE3) suşunda DsbA, MBP sinyal peptitleri kullanarak yapılan tasarımla periplazmik ve YebF sinyal peptidi kullanarak yapılan tasarımla hücre dışında üretimleri karşılaştırılmıştır. Üretim sonrası >99% oranında saflaştırılan scFv’lerin, çözünür ve aktif formda oldukları yapılan çalışmada gösterilmiştir. Sıcaklık taraması yapılarak, üretim için uygun sıcaklık belirlenmiştir. Üretim sürecinin optimize edilmesinin ardından scFv’ler farklı yönleri ile kıyaslanmıştır. Özgüllükleri değerlendirilen periplazmik, sitoplazmik ve ekstraselüler örnekler, in vitro analizlerde antijene doğru şekilde bağlanmıştır. Sitoplazmik örneğin üretim verimi 6,2 mg/L iken, spMBP ile yapılan periplazmik üretimin verimi 5,1 mg/L, hücre dışı üretim verimi ise 2,5 mg/L olarak bulunmuştur. Ayrıca, özgül ısı ve erime sıcaklıklarına (Tm) bakıldığında, sitoplazmik örneğin termal olarak daha kararlı olduğu sonucuna ulaşılmıştır. Özetle bu çalışma, tıbbi tanı ve tedavide rol oynayan, rekombinant DNA teknolojisi ile üretilecek ve disülfit bağı içeren proteinlerin üretim maliyetlerini azaltım sürecine rehberlik edebilecek bir çalışma olmuştur. Özellikle anti-HER2 scFv’nin aktif ve çözünür formdaki üretimi, kompleks proteinlere özgü yöntemler ve suşlar kullanarak E. coli’de klonlanması ve farklı yönleriyle değerlendirilmesi literatüre önemli bir katkı sağlayacaktır.tr_TR
dc.contributor.departmentBiyomühendisliktr_TR
dc.embargo.termsAcik erisimtr_TR
dc.embargo.lift2023-06-05T11:29:03Z
dc.fundingBilimsel Araştırma Projeleri KBtr_TR


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