Productıon of Recombınant Antıbody Fragments in Bacterıa and Theır Development Towards Medıcal Dıagnosıs
Özet
Antibodies and antibody derivatives have been widely used in diagnostic and therapeutic applications as well as in biotechnological research tools. Since their application in these areas requires for their production with a high yield, recombinant protein technology has been used to meet the increasing demand in the global market and to reduce production costs. Single-chain variable fragment (scFv), which is one of the most widely used recombinant antibody fragments, is formed by linking heavy and light units of variable domains with a flexible linker peptide. Successful construction of scFvs is based on composition, length and flexibility of linker peptides in addition to variable domain order.
In this thesis, the production of model scFvs, that are highly soluble, active and correctly folded in Escherichia coli was proposed and the effects of linker peptides to properties of scFvs towards immobilization for diagnostic applications were investigated. For this purpose, anti-HER2 scFv against human epidermal growth receptor 2 (HER2) and scFv13R4 against β-galactosidase were selected as model antibody fragments. Firstly, scFv version of the full-length antibody targeting HER2 glycoprotein, which overexpresses in breast cancer patients, was constructed via splicing by overlap extension polymerase chain reaction (SOE-PCR). Anti-HER2 scFv was expressed in the cytoplasm of E. coli strains BL21(DE3) and SHuffle T7 Express cells, the latter of which has more oxidizing conditions for disulfide bond formation, to determine the suitable strain for functional production of the scFv. Evaluation of the host strains revealed significantly higher solubility and up to four-fold higher antigen-binding activity of anti-HER2 scFv expressed in SHuffle T7 Express versus BL21(DE3). Hence, subsequent productions of scFvs were performed in SHuffle T7 Express. Thereafter, anti-HER2 scFvs were designed in two different domain orientations, which were VH-linker-VL or VL-linker-VH, to determine the optimum domain orientation for this scFv. They were expressed in SHuffle cells to compare their properties in terms of solubility, monomer/dimer formation, binding activity and affinity. Soluble production was characterized by Western blot analysis, and in vitro immunological characterization of anti-HER2-scFv was performed with enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Consequently, domain orientation of anti-HER2 scFv was investigated for the first time, and each scFv showed high binding activity and selectivity against HER2, regardless of the variable domain orientation. In addition, effects of bioprocess operation parameters such as induction temperature, inducer concentration, duration of induction and medium composition on expression of scFvs were investigated. It was found that IPTG concentration did not significantly affect the anti-HER2 scFv expression, higher amount of soluble anti-HER2 scFvs were observed between 16-20 h after induction and ratio of soluble expression increased at temperatures lower than 30°C. Overexpression of anti-HER2 scFv was also observed when produced in chemically defined minimal medium in SHuffle cells. In the second part of the study, anti-HER2 scFv in VL-linker-VH orientation was used, owing to its relatively higher expression. ScFvs were re-designed by adding linkers with different amino acid composition, towards immobilization onto polystyrene (PS) surfaces and characteristics of the new scFvs were investigated. Direct, indirect and sandwich ELISA was performed for determination of immobilization, antigen-binding properties and detection of HER2, respectively. It was observed that the immobilization activities of scFvs with different linkers changed in relation to PS plate type, and absence or presence of Tween 20 in coating buffer. Additionally, the same linkers were cloned into the other model antibody fragment, scFv13R4, to investigate the versatility of the effects of linkers and similar results were observed. Lastly, in silico analysis was performed to observe the effect of linkers on stability and functionality of anti-HER2 scFvs with homology modelling and molecular dynamics (MD) simulation. Based on the experimental results and MD simulations, one of the designed linkers was found to be an appropriate substitution for the standard linker within the scFvs for immunoassay applications. Overall, the model scFvs constructed in this study hold great potential as biorecognition elements in immunoassays or diagnostic tools owing to their highly soluble expression and biologically active forms with nM range dissociation constants (Kd).
