Anti-Her2 SCFV Antikor Fragmanının Escherichia coli Bakterisinde Üretim Bölgesinin Optimizasyonu

Abstract

Antibodies 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.