Recombinant single-chain antibody fragment (scFv) is a genetic fusion of the variable domain of the light chain of IgG antibodies (VL) and the variable domain of the heavy chain of IgG antibodies (VH). Their molecular mass varies from 25 to 30 kDa, providing them with certain advantages that make them useful for disease treatment and diagnosis. Among these is amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that affects upper and lower motor neurons. Among the proteins associated with ALS are fused in sarcoma (FUS) and matrin 3 (MATR3). Mutations in FUS and MATR3 genes, among others, disrupt protein transport between the nucleus and cytoplasm, leading to the formation of protein aggregates in the cytoplasm. The development of ALS is also influenced by aminoacyl-tRNA synthetases (aaRS), key enzymes in the translation process. As part of the thesis project, we aimed to express scFv against proteins involved in ALS, specifically Z-FUS-5 and Z-MATR3-4, and scFv against aminoacyl-tRNA synthetases, O-LARS-15 and J-HARS-4, using the E. coli expression system. First competent E. coli BL21[DE3] cells were transformed with vectors. Expression was then induced with the addition of IPTG, and scFvs were isolated from the periplasm, as they contained a signal sequence from outer membrane protein A (ompA). Since all scFvs contained a hexahistidine tag, nickel affinity chromatography was used for isolation. After isolation, scFvs Z-FUS-5 and Z-MATR3-4 were further characterized using dot blotting. We found that the mentioned scFvs recognize the proteins FUS and MATR3 in denatured and native forms.
|
