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.
Hashimoto
Y
Y
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Ikenaga
T
T
Tanigawa
K
K
Ueda
T
T
Ezak
I
I
Imoto
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology*; D009838:Oligodeoxyribonucleotides; D017510:Protein Folding; D012217:Rheumatoid Factor / Q000737:chemistry / Q000276:immunology*
D007128:Immunoglobulin Fragments; D009838:Oligodeoxyribonucleotides; D012217:Rheumatoid Factor