Bacterial expression and refolding of single-chain Fv fragments with C-terminal cysteines.
Two antibody single-chain Fv (scFv) fragments carrying five C-terminal histidine residues were expressed in Escherichia coli as periplasmic inclusion bodies. Their variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody 215 (MAb215), specific for the largest subunit of RNA polymerase II of Drosophila melanogaster and rat MAb Yol1/34, specific for pig brain alpha-tubulin. ScFv-215 contains an additional cysteine residue near to its C-terminus. After solubilization of inclusion bodies followed by immobilized metal affinity chromatography (IMAC) in 6M urea and a renaturation procedure, scFv monomers, noncovalent dimers, and aggregated antibody fragments were separated by size exclusion chromatography. In addition, a fraction of disulfide-bonded scFv-215 homodimers (scFv')2 was also isolated. The various antibody forms appear to be in equilibrium after renaturation since first peak composed mainly of aggregates could be resolved into a similar pattern of aggregates, dimers, and monomers after repeating the denaturation/renaturation procedure. All fractions of the recombinant scFv-215 demonstrated high antigen-binding activity and specificity as shown by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv')2 have binding constants quite close to those of the parental MAbs and fourfold higher than scFv' monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.
Recombinant Antibody Research Group (FSP 4/0445), German Cancer Research Center, Heidelberg, Germany.
S M
SM
Kipriyanov
S
S
Dübel
F
F
Breitling
R E
RE
Kontermann
S
S
Heymann
M
M
Little
D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D017510:Protein Folding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D003545:Cysteine