Enhanced Formation of Disulfide-bridged Dimer (Fab-PE38)2 Utilizing Repeats of the Fab Binding Domain of Protein G

Abstract

Fab-PE38 used in this study is B3(Fab)-ext-PE38, and it is an antibody toxin that is made by fusing theAntibody toxins are made by fusing toxins to specific antibodies that selectively bind to cancer cells (1). (B3(Fab)-ext-PE38) 2 is the disulfide-bridged dimer of monomer B3(Fab)-ext-PE38 (2). We used the B3 monoclonal antibody as a model antibody, and it binds to a carbohydrate antigen found on many carcinomas (3, 4). PE38 is a 38-kDa derivative of Pseudomonas exotoxin (PE) 2 that catalyzes ADP-ribosylation of protein elongation factor 2 and inactivates it (5, 6). PE38 is a truncated form of the polypeptide that cannot bind to target cells by itself.B3(Fab)-ext-PE38 contains the light chain (V L -C L ) and the long chain in which the Fd chain (V H -C H1 ) is fused to PE38. The ext connects the Fd chain and PE38, producing the long Fd(V H -C H1 )-ext-PE38 chain. The Fab domain is formed by the formation of an interchain disulfide bond between the light chain and Fd chain. Two cysteines for this interchain disulfide bond are the one located at the end of C L domain of the light chain and the other one at the end of the C H1 domain of the long chain. The Fab toxin has two advantages over the single chain Fv(scFv) toxin in which two variable domains (V H and V L ) of antibody are connected with flexible linker. First, the refolding yield of Fab toxin is 10-fold higher than that of scFv toxin (7,8). Second, the stability of the Fab antibody toxin is higher than that of scFv toxin (9, 10).(B3(Fab)-ext-PE38) 2 has higher cytotoxicity than the monomeric B3(scFv)-PE38. Specifically, it has about 11-fold higher cytotoxicity in the CRL-1739 cell line than that of B3(scFv)-PE38 (2). The dimer is produced by the formation of a disulfide bond bridge between two monomers with the Cys residue located in the ext region in the Fd(V H -C H1 )-ext-PE38 chain. This disulfide bond is formed by the random collision of two Cys residues in two monomers. In our previous report, the refolding yield of the dimer was 0.06% (2). This is very low when compared with the yield of the monomer, which was ϳ10%.A dimeric antibody toxin is predicted to have several advantages over monomeric antibody toxins. The existence of two antigen binding domains should confer an increase in binding avidity. A dimeric antibody toxin will deliver two toxins at the same time to target cells, which enables it to kill target cells with a higher cytotoxicity than monomeric antibody toxin. Dimeric antibody toxin should also be more stable than scFv toxin.Streptococcal protein G is known to bind to IgG. It has a serum albumin binding domain and three IgG binding domains. It has been identified that the third IgG binding domain (domain III) of Streptococcal protein G binds to the Fab fragment of IgG (11).In this study, we focused on producing the dimer from the monomers that had already been refolded and purified. We constructed recombinant tandem repeats of Streptococcal protein G domain III and used it to make complexes that have multiple monomers bound. We found...

Preparation and mechanical characterisation of giant unilamellar vesicles by a microfluidic method

“…This is because very minor changes in the mechanical properties are critical to a wide range of general and highly specific functions of the cell, for example, it has been suggested that deformations in the plasma membrane control the gating mechanism of mechanosensitive channel proteins. 10 The mechanical properties also impact the binding of specific proteins to the membrane bilayer. The torque tension and spontaneous elastic curvature of the bilayer have both been found to affect the degree of protein binding in the membrane.…”

Title: Supercomplex formed by the cross-binding of multiple antibody-single repeat chain complex gives signal amplification of antibody based assay

“…The domain III of the three Ig binding domains from protein G was used to construct up to 10 repeat (TR10) (Supplementary Fig. 1) 5 . As expected from its antibody binding characteristics, we found that TR10 could bind up to three Fab containing molecules.…”

“…were prepared according to the method described previously 5 . Primary Antibody and secondary antibody were purchased from Santa cruz biotech, which are mouse anti-beta-actin monoclonal antibody (sc-47778) and horse radish peroxidase conjugated goat anti-mouse antibody.…”