Production of functional single-chain variable fragment against Tumor Necrosis Factor alpha in engineered Escherichia coli.

Abstract

Human TNF-alpha is a non-glycosylated protein of 17 kDa molecular weight and trimeric in vivo. Low levels of human TNFα aid in maintaining homeostasis and promoting the replacement of injured tissue, however, high levels of human TNFα cause autoimmune diseases such as reumatoid arthritis. To regulate the level of h-TNFα, many therapeutic antibodies for target h-TNFα have been produced. Production of small sized recombinant antibodies such as single-chain variable fragment (scFv) would be more effective than production of monoclonal antibodies. ScFv is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. In previous research, genes for the scFv of a monoclonal antibody (mAb) against TNF-α were cloned and expressed in Escherichia coli. However, the scFv was expressed in insoluble form, so the purified scFv was refolded in vitro to acquire soluble and active scFv. To produce soluble scFv against human TNF-α in recombinant E. coli C41(DE3), the expression vector for the scFv against TNFα fused with the maltose binding protein (MBP) was applied. The vector was constructed and transformed into E. coli C41(DE3). The resulting strain was able to express the scFv successfully in soluble form. Second, the scFv and MBP fusion protein was purified by affinity chromatography using MBP as a ligand and the binding activity of the purified scFv against TNFα was verified by using Biacore T100. The association constant (ka) of 2040 (1/M•s) and dissociation constant (kd) of 0.001769 (1/s) were estimated. Finally, the KD value defined as the equilibrium dissociation constant between the antibody and its antigen was determined to 8.671E-7 (M). The lower KD value represents that the antibody has the higher affinity to an antigen. As typical antibodies have the KD value of 10-7~10-8 (M). The biological function of the scFv tw TNFα produced in E. coli could be confirmed. Third, production of anti-TNFα scFv by fed-batch fermentation of engineered E. coli was attempted to produce 72.7 mg/L. In conclusion, this thesis has demonstrated production of the functional scFv against human TNFα in recombinant E. coli for diagnosis and therapeutic applications.