Bivalent Fv antibody fragments obtained by substituting the constant domains of a fab fragment with heterotetrameric molybdopterin synthase.
The antibody Fv fragment is the smallest functional unit of an antibody but for practical use, the VH/VL interface requires stabilization, which is usually accomplished by a peptide linker that joins the two variable domains to form a single chain Fv fragment (scFv). An alternative format to scFv is proposed that (i) allows stabilization of the Fv fragment, and (ii) restores the bivalency of the antibody as a pseudo-F(ab')2 format. This new antibody fragment was constructed by replacing the CHI and CL domains of the Fab fragment with heterotetrameric molybdopterin synthase (MPTS). We found that this format, named MoaFv, improved significantly the cytoplasmic expression of the Fv as a soluble protein in BL21 or Origami Escherichia coli strains. This MoaFv format is expressed as a homogeneous heterotetrameric protein with a Mr value of 110 kDa containing two functional binding sites as revealed by active site titration. In its native condition at 37 degrees C or in the presence of urea, this format was nearly as stable as the corresponding scFv, indicating that non-covalent interactions between the MPTS subunits can replace the covalent peptide linker in scFv. Finally, this MoaFv construct could be a useful format when bivalency is desirable to improve the functional avidity.
UMR-CNRS n degrees 6204, Biotechnoligie, Biocatalyse et Bioréegulation, Nates cedex, France.
Kliment
K
Petrov
Michel
M
Dion
Lionel
L
Hoffmann
Thierry
T
Dintinger
Alain
A
Defontaine
Charles
C
Tellier
D001483:Base Sequence; D001704:Biopolymers; D002850:Chromatography, Gel; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013466:Sulfurtransferases / Q000737:chemistry / Q000378:metabolism*
D001704:Biopolymers; D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D013466:Sulfurtransferases; C085585:molybdopterin synthase