Increase of spacer sequence yields higher dimer (Fab-spacer-toxin)2 formation

Meehyeon Yoo, Jaeseon Won, Yongchan Lee, Muhyeon Choe

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)


The divalent antibody-toxins are expected to have increased binding avidities to target cells because of the two cell-binding domains. However, previous studies showed that the refolding yield of divalent antibody-toxin is very low, and it is assumed that homodimer formation of antibody-toxin is strongly interfered by the repulsion between the two large toxin domains that come close to each other during dimer formation. In this study, B3 antibody was used as a model antibody, and its Fab domain was used to construct three different kinds of Fab divalent molecules, [B3(Fab)-toxin]2. The monomer Fab-toxin molecules were made by fusing the Fab domain of monoclonal antibody B3 to PE38, a truncated mutant form of Pseudomonas exotoxin (PE), and a connecting sequence that contained spacer amino acid sequence (G4S)n (n=1, 2, 3) was inserted between Fab and PE38. The prepared divalent molecules were [Fab-S1, 2, 3-PE38]2 (=[Fab-SKPCIST-KAS(G4S)nGGPE-PE38]2 (n=1, 2, 3)), and they are derivatives of previously studied [Fab-H2cys-PE38]2 (=[Fab-SKPCIST-KASGGPE-PE38]2). In [Fab-S1, 2, 3-PE38]2, two Fab-S1, 2, 3-PE38 monomers were covalently linked by the disulfide bond bridge made from cysteine in the -SKPCIST-sequence. The insertion of spacer amino acids after the disulfide bridge resulted in a 12-18 fold higher yield of dimer formation than previously constructed [Fab-H1cys-PZ38]2 [7], 3-4-fold higher than [Fab-ext-PZ38]2 [25]. These two molecules have less amino acid spacer sequence between the disulfide bridge and PE38 domain. The design of [Fab-PE38]2 in this study gave molecules with a higher refolding yield. The results of cytotoxicity assay showed a higher cytotoxic effect of these divalent molecules than that of the monovalent scFv-PE38 molecule.

Original languageEnglish
Pages (from-to)1097-1103
Number of pages7
JournalJournal of microbiology and biotechnology
Issue number7
Publication statusPublished - 2006 Jul


  • B3 antibody
  • Cytotoxicity
  • Divalent (Fab-toxin)
  • Divalent immunotoxin
  • Pseudomonas exotoxin A
  • Recombinant antibody refolding

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology


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