Abstract
Depending on the linker length between the V and the V domain, single-chain Fv (scFv) antibody fragments form monomers, dimers (diabodies) or higher oligomers. We aimed at generating a diabody of the anti-MET antibody 3H3 to use it as crystallization chaperone to promote crystallization of the MET ectodomain through the introduction of a pre-formed twofold axis of symmetry. Size exclusion chromatography, however, suggested the protein to be monomeric. Hence, we used scattering techniques applied to solutions to further investigate its oligomerization state. The small angle X-ray scattering (SAXS) curve measured for our protein nicely fits to the scattering curve calculated from the known crystal structure of a diabody. In addition, concentration-dependent photon correlation spectroscopy (PCS) measurements revealed a hydrodynamic radius of 3.4 nm at infinite dilution and a negative interaction parameter , indicating attractive interactions that are beneficial for crystallization. Both SAXS and PCS measurements clearly suggest our antibody fragment to be a diabody in solution. Chemical cross-linking with glutaraldehyde and cell motility assays confirmed this conclusion.
Highlights
Antibodies belong to the most variable proteins of higher organisms
P. pastoris was stably transfected with the desired target gene and the protein was expressed for five days in minimal medium [41]
In this work we have studied the solution structure of the IgG fragment 3H3
Summary
Antibodies belong to the most variable proteins of higher organisms. This variability is necessary to recognize a vast number of different pathogens or foreign molecules and to trigger an adequate immune response. There are five different classes of human antibodies (IgA, IgD, IgE, IgG, IgM) with specific tasks [1,2]. Its overall structure resembles a Y shape with one fragment crystallizable (Fc) and two antigen binding fragments (Fab). The Fc part is responsible for the stability of the antibody and for the recruitment of effector molecules. The antigen binding sites are located at the ends of the Fab arms, in the variable region of the heavy (VH ) and the light chains (VL ) [3,4]
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