To check the formation of complexes between monomers and protein G domain III tandem repeat, we carried out association reactions using tandem repeats and B3(Fab)-ext-PE38 in different combinations and analyzed the complexes, and the yields of the formation of the disulfide bond-bridged dimer were evaluated. == EXPERIMENTAL PROCEDURES == == == == == == Preparation of Tandem Repeats of Streptococcal Protein G Domain III Amifostine == Protein G domain III was cloned by PCR fromStreptococcuschromosomal DNA (Korean Collection for Type Cultures (KCTC) strain number 3098), which was obtained from the KCTC. of it greatly enhances the yield of the disulfide-bridged dimer. Keywords:Antibodies, Disulfide, Fusion Protein, Protein Cross-linking, Protein Folding, Fab Binding Domain, Antibody Toxin, Disulfide-bridged Dimer, Protein G, Tandem Repeat == Introduction == Antibody toxins are made by fusing toxins to specific antibodies that selectively bind to cancer cells (1). (B3(Fab)-ext-PE38)2is Amifostine 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 ofPseudomonasexotoxin (PE)2that 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 (VL-CL) and the long chain in which the Fd chain (VH-CH1) is fused to PE38. The ext connects the Fd chain and PE38, producing the long Fd(VH-CH1)-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 CLdomain of the light chain and the other one at the end of the CH1domain of the long chain. The Fab toxin has two advantages over the single chain Fv(scFv) toxin in which two variable domains (VHand VL) 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)2has 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(VH-CH1)-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 10%. 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 Amifostine 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. Streptococcalprotein G is known to bind to IgG. Amifostine It has a serum albumin binding domain and three IgG binding domains. It has been identified that the third IgG binding domain (domain III) ofStreptococcalprotein 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 ofStreptococcalprotein G domain III and used Amifostine it to make complexes that have multiple monomers bound. We found that the monomers bound to a tandem repeat are in close proximity enough to result in an increased formation of a disulfide bond bridge COPB2 between two monomers, producing the dimer in higher yield. To check the formation of complexes between monomers and protein G domain III tandem repeat, we carried out association reactions using tandem repeats and B3(Fab)-ext-PE38 in different combinations and analyzed the complexes, and the yields of the formation of the disulfide bond-bridged dimer were evaluated. == EXPERIMENTAL PROCEDURES == == == == == == Preparation of Tandem Repeats of Streptococcal Protein G Domain III == Protein G domain III was cloned by PCR fromStreptococcuschromosomal DNA (Korean Collection for Type Cultures (KCTC) strain number 3098), which was obtained from the KCTC. The following primers were used for the PCR reaction: P1 (5-GGGCAT.