Expression and characterization of human rheumatoid factor single-chain Fv

The variable region of heavy chain [V(H)] of human rheumatoid factor (hRF) IgM was connected with the variable region of light chain [V(L)] with the peptide-linker (GGGSGGGSGGGS) by genetic engineering method and the single-chain Fv (scFv) was expressed in E. coli. On design, scFv and scFv (tag) were planned; the latter had a detection marker at the carboxyl-terminal. These scFvs were expressed as inclusion bodies in E. coli, purified in the presence of 8 M urea by gel filtration and renatured to the active form in vitro. As a control, the Fv, non-covalently associated V(H) and V(L) fragments, was also constructed. The 3 derivatives showed almost the same binding activity to rabbit-IgG to which hRF is cross-reactive. ScFv (tag) was the most stable against urea among the 3 derivatives.