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

• Published in: The Journal of biological chemistry Vol. 285; no. 8; pp. 5127 - 5131
• Main Authors: Lee, YongChan, Park, JongChan, Kweon, SoonWook, Choe, MuHyeon
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 19-02-2010; American Society for Biochemistry and Molecular Biology
• Subjects: ADP Ribose Transferases - chemistry; Animals; Antibodies; Antibodies, Neoplasm - chemistry; Antibody Toxin; Bacterial Toxins - chemistry; Disulfide; Disulfide-bridged Dimer; Disulfides - chemistry; Exotoxins - chemistry; Fab Binding Domain; Fusion Protein; Humans; Immunoglobulin Fab Fragments - chemistry; Immunotoxins - chemistry; Nerve Tissue Proteins - chemistry; Protein Cross-linking; Protein Folding; Protein G; Protein Multimerization; Pseudomonas aeruginosa Exotoxin A; Single-Chain Antibodies - chemistry; Tandem Repeat; Virulence Factors - chemistry; Fusion Protein; Fab Binding Domain; Protein G; Disulfide; Antibodies; Tandem Repeat; Protein Folding; Protein Cross-linking; Antibody Toxin; Disulfide-bridged Dimer
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.C109.006395

Fab-PE38 used in this study is B3(Fab)-ext-PE38, and it is an antibody toxin that is made by fusing the Pseudomonas exotoxin to the Fab domain of B3 antibody. This antibody toxin selectively binds to cancer cells and kills the target cancer cells. B3(Fab)-ext-PE38 has a cysteine residue on the ext sequence, and (B3(Fab)-ext-PE38)2 is the disulfide-bridged dimer of the B3(Fab)-ext-PE38 monomer. (B3(Fab)-ext-PE38)2 has been found to have 11-fold higher cytotoxicity on the CRL-1739 cell line than monomeric B3(scFv)-PE38. We made a recombinant tandem repeat of the domain III of Streptococcal protein G that has Fab binding property up to seven repeats. Multiple monomers were found to form non-covalent complexes with this tandem repeat. Complexes were purified by size-exclusion chromatography, and we could enhance the production of the disulfide-bridged dimer by reduction and oxidation of the complexes. The tandem repeat makes close intermolecular interactions between monomers possible, and the use of it greatly enhances the yield of the disulfide-bridged dimer.