• Journal of Microbiology and Biotechnology
• /
• v.16 no.7
• /
• pp.1104-1110
• /
• 2006

Recombinant antibody-toxin is a bifunctional protein that binds and kills a target cell expressing a specific antigen on the surface of the cell, and its structure is chimeric, in which a toxin is fused to an antigen-binding domain such as scFv or Fab. Divalent antibody-toxin molecules showed higher cytotoxicities against cancer cell lines than monovalent molecules. However, the yields of the divalent molecules were very low. In this study, we introduced the CH2, CH3, or CH2-CH3 (=Fc) domain of antibody in the middle of the Fab-toxin between the hinge region of human IgG1 and the toxin domain to increase the yield. The covalently bonded dimer could be formed by three disulfide bridges from cysteine residues in the hinge region. The molecule with the CH3 domain showed about 3-fold higher dimerization yield than previously constructed Fab-toxin molecules, while maintaining the cytotoxic activity comparable to that of scFv-toxin. However, the introduction of CH2 or Fc domain to the same position showed little effect on the dimerization yield. We also observed that the introduction of the CH3 region made it possible to form noncovalently associated dimer molecules.

• Bulletin of the Korean Chemical Society
• /
• v.30 no.5
• /
• pp.1101-1106
• /
• 2009

Multivalent and multi-specific antibodies can provide valuable tools for bio-medical research, diagnosis and therapy. In antigen-antibody interactions, the avidity of antibodies depends on the affinity and the number of binding sites.$^1$ As artificial multivalent antibody agents, single chain Fv-streptavidin fusion tetramer proteins $(scFv-SA)_4$ have been previously tested.$^{1,\;2}$ Although, the Fab domain is known to be more stable than scFv in animal models,$^{3,\;4}$ it has never been used to make a multivalent agent with a streptavidin fusion. In this study, we prepared tetra-valent $(Fab-cSA)_4$ by fusing Fab with core streptavidin (cSA). This molecule was made using inclusion body production, refolding and chromatography purification. Affinities of the Fab-cSA tetramer and a scFv-cSA tetramer to a cell surface antigen were compared by ELISA using biotin-HRP. The Fab-cSA tetramer showed higher binding avidity than the scFv-cSA tetramer. The higher binding avidity of the Fab-cSA tetramer demonstrates its potential as a therapeutic agent for target-specific antibody therapy.

• Journal of Microbiology and Biotechnology
• /
• v.16 no.7
• /
• pp.1097-1103
• /
• 2006

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=l, 2, 3) was inserted between Fab and PE38. The prepared divalent molecules were $[Fab-S\;1,\;2,\;3-PE38]_{2}\;(=[Fab-SKPCIST-KAS(G_{4}S)nGGPE-PE38]_{2}\;(n=1,\;2,\;3))$, and they are derivatives of previously studied $[Fab-H2cys-PE38]_{2}\;(=[Fab-SKPCIST-KASGGPE-PE38]_{2})$. In $[Fab-Sl,\;2,\;3-PE38]_{2}$, 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 $[Fab-Hlcys-PZ38]_{2}[7]$, 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.

• Bulletin of the Korean Chemical Society
• /
• v.22 no.12
• /
• pp.1361-1365
• /
• 2001

Recombinant immunotoxins are hybrid cytotoxic proteins designed to selectively kill cancer cells. A divalent immunotoxins, [B3(FabH1)-PE38]2, was constructed by recombining Fab domain of B3 antibody as a cell-targeting domain and Pseudo monas exotoxin A (PE) as a cytotoxic domain. Monoclonal antibody, B3, is the murine antibody (IgG1k) directed against Lewis Y-related carbohydrate antigens, which are abundant on the surface of many carcinomas. Fab fragment of this antibody was used in this study with the modified hinge sequence where last two cysteines out of three were mutated to serine. PE is a 66 kDa bacterial toxin that kills eukaryotic cells by inhibiting protein synthesis with ADP ribosylation of ribosomal elongation factor 2 (EF2). Fc region of B3 antibody was substituted with the truncated form of PE (38 kDa, PE38) on DNA level. [B3(FabH1)-PE38]2 was formed by disulfide bond between cysteines in the modified hinge region of B3(FabH1)-PE38. Each polypeptide for recombinant immunotoxins was overexpressed in Escherichia coli and collected as inclusion bodies. Each inclusion body was solubilized and refolded, and cytotoxic effects were measured. Divalent immunotoxins, [B3(FabH1)-PE38]2, had ID50 values of about 10 ng/mL on A431 cell lines and about 4 ng/mL on CRL1739 cell lines. Control immunotoxins, B3(scFv)-PE40, had ID50 values of about 28 ng/mL on A431 cell lines and about 41 ng/mL on CRL1739 cell lines. Divalent immunotoxins, [B3(FabH1)-PE38]2, had higher cytotoxic effects than B3(scFv)-PE40 control immunotoxins.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.7 no.2
• /
• pp.127-131
• /
• 2003

Protein disulfide isomerase (PDI) found in the endoplasmic reticulum (ER) catalyzes disulfide bond exchange and assists in protein folding of newly synthesized proteins. PDI also functions as a molecular chaperone and has been found to be associated with proteins in the ER. In addition, PDI functions as a subunit of two more complex enzyme systems: the prolyl-4-hydroxylase and the triacylglycerol transfer proteins. A cDNA that encodes protein disulfide isomerase was previously isolated from Bombyx mori (bPDI), in which open reading frame of 494 amino acids contained two PDI-typical thioredoxin active site of WCGHCK and an ER retention signal of the KDEL motif at its C-terminal, and we report its functional characterization here. This putative bPDI cDNA is expressed in insect Sf9 cells as a recombinant proteins using baculovirus expression vector system. The bPDI recombinant proteins are successfully recognized by antirat PDI antibody, and shown to be biologically active in vitro by mediating the oxidative refolding of reduced and scrambled RNase. This suggests that bPDI may play an important role in protein folding mechanism of insects.

• Microbiology and Biotechnology Letters
• /
• v.26 no.1
• /
• pp.15-22
• /
• 1998

The dnrF gene, responsible for conversion of aklavinone to $\varepsilon$-rhodomycinone via C-11 hydroxylation, was mapped in the daunorubicin gene cluster of Streptomyces peucetius subsp. caesius ATCC 27952, close to drrAB, one of the anthracycline resistance genes. To characterize the enzymatic properties of the aklavinone 11-hydroxylase, the dnrF gene was overexpressed in Escherchia coli. The pET-22(+) plasmid which has the T7 promoter under the control of lacUV5 gene was used for the overexpression of the dnrF gene, and the recombinant plasmid pET213 that contains the dnrF gene linked to the T7 promoter of pET-22b(+) was introduced into the E. coli BL2l. When the expression of the dnrF gene was induced by IPTG at the final concentration of 1 mM, the induced protein could be detected in SDS-PAGE only in insoluble precipitate. The insoluble protein was electroeluted from the gel and used for the preparation of antiserum in mice. Various culture conditions were tested to maximize the expression of the aklavinone 11-hydroxylase in soluble form. The enzymatic activity was checked by the bioconversion experiment, and the protein was confirmed by the SDS-PAGE and the Western blot analysis. From the analysis of the data, it was concluded that the culture induced with IPTG at the final concentration of 0.02 mM at 37$^{\circ}C$ yielded the best productivity of active form of enzyme.