Disulfide bond bridged divalent antibody-toxin, (Fab-PE38fl)(2), with the toxin PE38 fused to the light
- Authors
- Won, JaeSeon; Choe, MuHyeon
- Issue Date
- Aug-2008
- Publisher
- KOREAN SOC MICROBIOLOGY & BIOTECHNOLOGY
- Keywords
- antibody refolding; light chain-toxin fusion; divalent antibody-toxin; homodimer; cytotoxicity; Pseudomonas exotoxin A
- Citation
- JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, v.18, no.8, pp.1475 - 1481
- Indexed
- SCIE
SCOPUS
KCI
- Journal Title
- JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
- Volume
- 18
- Number
- 8
- Start Page
- 1475
- End Page
- 1481
- URI
- https://scholar.korea.ac.kr/handle/2021.sw.korea/122919
- ISSN
- 1017-7825
- Abstract
- B3 antibody specifically binds the Lewis(Y)-related carbohydrate antigen of man), carcinomas, and it is used as a model antibody in this study. In a previous study, the Fab fragment of the antibody was fused to a 38 kDa truncated form of Pseudomonas exotoxin A, PE38, to make Fab-PE38, where PE38 is fused to the Fd fragment of the Fab domain. This parent monomer molecule, Fab-PE38, had no cysteine in the hinge region, and it could not make a disulfide bond to form a disulfide bond bridged homodimer [7]. In this study, we constructed three different kinds of divalent Fab-toxin fusion homodimers where the toxin is fused to the light chain of Fab, (Fab-PE38fl)(2). In addition to the PE38 toxin fused to the light chain, these three molecules have different hinge sequences h1, h2, and h3 making Fabh1-, Fabh2-, and Fabh3-PE38fl monomers, respectively. These hinges contain only one cysteine on different positions of the hinge sequence. The disulfide bond between the hinge region of two monomers forms homodimers (Fabh1-PE38fl)(2), (Fabh2-PE38fl)(2), and (Fabh3-PE38fl)(2). The refolding yields of these dimers were 516-fold higher than a previously constructed dimer where the PE38 was fused to the Fd fragment (Fabh1-PE38)(2) [8]. Our data suggest that the steric repulsion between the two PE38s in (Fabh1-PE38)(2) during disulfide bridge formation is relieved by fusing it at the end of the light chain. The best cytotoxicity value of these dimers; showed about 2.5-fold higher on an MCF7 cell line than that of the monovalent reference molecule in ng/ml scale, which is 15-fold higher in pM scale.
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