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A novel screening system for claudin binder using baculoviral display.

Kakutani H, Takahashi A, Kondoh M, Saito Y, Yamaura T, Sakihama T, Hamakubo T, Yagi K - PLoS ONE (2011)

Bottom Line: CL4-displaying BV interacted with a CL4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), but it did not interact with C-CPE that was mutated in its CL4-binding region.C-CPE did not interact with BV and CL1-displaying BV.We screened the library for CL4 binders by affinity to CL4-displaying BV, and we found that the novel CL4 binders modulated the tight-junction barrier.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bio-Functional Molecular Chemistry, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan.

ABSTRACT
Recent progress in cell biology has provided new insight into the claudin (CL) family of integral membrane proteins, which contains more than 20 members, as a target for pharmaceutical therapy. Few ligands for CL have been identified because it is difficult to prepare CL in an intact form. In the present study, we developed a method to screen for CL binders by using the budded baculovirus (BV) display system. CL4-displaying BV interacted with a CL4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), but it did not interact with C-CPE that was mutated in its CL4-binding region. C-CPE did not interact with BV and CL1-displaying BV. We used CL4-displaying BV to select CL4-binding phage in a mixture of a scFv-phage and C-CPE-phage. The percentage of C-CPE-phage in the phage mixture increased from 16.7% before selection to 92% after selection, indicating that CL-displaying BV may be useful for the selection of CL binders. We prepared a C-CPE phage library by mutating the functional amino acids. We screened the library for CL4 binders by affinity to CL4-displaying BV, and we found that the novel CL4 binders modulated the tight-junction barrier. These findings indicate that the CL-displaying BV system may be a promising method to produce a novel CL binder and modulator.

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Screening of a novel CL4 binder.A) Enrichment of phages with affinity to CL4-BV. CL4-BVs coated on immunotubes were incubated with the C-CPE-derivative phage library at 1.6×1012 CFU titer (1st input phage). The phages bound to CL4-BV were recovered (1st output phage). The CL4-BV-binding phages were subjected to two additional cycles of the incubation and wash step, resulting in 2nd, 3rd output phage. The ratio of output phage to input phage titers was calculated. B) Monoclonal analysis of C-CPE-derivative phage. CL4-BV-bound phage clones were isolated from the 2nd and 3rd output phages, and the interaction of the monoclonal phage with CL4-BV was examined by ELISA with anti-M13 antibody as described in Materials and methods. Data are expressed as relative binding to that of C-CPE-phage indicated by the most right column.
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pone-0016611-g003: Screening of a novel CL4 binder.A) Enrichment of phages with affinity to CL4-BV. CL4-BVs coated on immunotubes were incubated with the C-CPE-derivative phage library at 1.6×1012 CFU titer (1st input phage). The phages bound to CL4-BV were recovered (1st output phage). The CL4-BV-binding phages were subjected to two additional cycles of the incubation and wash step, resulting in 2nd, 3rd output phage. The ratio of output phage to input phage titers was calculated. B) Monoclonal analysis of C-CPE-derivative phage. CL4-BV-bound phage clones were isolated from the 2nd and 3rd output phages, and the interaction of the monoclonal phage with CL4-BV was examined by ELISA with anti-M13 antibody as described in Materials and methods. Data are expressed as relative binding to that of C-CPE-phage indicated by the most right column.

Mentions: Then, we screened the CL4-binding phage by their affinity to CL4-BV. After addition of the C-CPE library to CL4-BV-adsorbed tubes, the CL4-BV-bound phages were recovered (1st screening). We repeated this screening process two more times (2nd screening and 3rd screening). If the number of CL4-bound phage is increased during the screening, the ratio of the incubated phage titers to the recovered phage titers will increase. As shown in Fig. 3A, the ratio was increased during screening from 4.5×10−7 to 5.5×10−5, indicating that the screening system for CL4 binders may work. Indeed, the number of monoclonal phage clones with high affinity to CL4-BV was increased after the 3rd screening compared with that after the 2nd screening (Fig. 3B).


A novel screening system for claudin binder using baculoviral display.

Kakutani H, Takahashi A, Kondoh M, Saito Y, Yamaura T, Sakihama T, Hamakubo T, Yagi K - PLoS ONE (2011)

Screening of a novel CL4 binder.A) Enrichment of phages with affinity to CL4-BV. CL4-BVs coated on immunotubes were incubated with the C-CPE-derivative phage library at 1.6×1012 CFU titer (1st input phage). The phages bound to CL4-BV were recovered (1st output phage). The CL4-BV-binding phages were subjected to two additional cycles of the incubation and wash step, resulting in 2nd, 3rd output phage. The ratio of output phage to input phage titers was calculated. B) Monoclonal analysis of C-CPE-derivative phage. CL4-BV-bound phage clones were isolated from the 2nd and 3rd output phages, and the interaction of the monoclonal phage with CL4-BV was examined by ELISA with anti-M13 antibody as described in Materials and methods. Data are expressed as relative binding to that of C-CPE-phage indicated by the most right column.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3038848&req=5

pone-0016611-g003: Screening of a novel CL4 binder.A) Enrichment of phages with affinity to CL4-BV. CL4-BVs coated on immunotubes were incubated with the C-CPE-derivative phage library at 1.6×1012 CFU titer (1st input phage). The phages bound to CL4-BV were recovered (1st output phage). The CL4-BV-binding phages were subjected to two additional cycles of the incubation and wash step, resulting in 2nd, 3rd output phage. The ratio of output phage to input phage titers was calculated. B) Monoclonal analysis of C-CPE-derivative phage. CL4-BV-bound phage clones were isolated from the 2nd and 3rd output phages, and the interaction of the monoclonal phage with CL4-BV was examined by ELISA with anti-M13 antibody as described in Materials and methods. Data are expressed as relative binding to that of C-CPE-phage indicated by the most right column.
Mentions: Then, we screened the CL4-binding phage by their affinity to CL4-BV. After addition of the C-CPE library to CL4-BV-adsorbed tubes, the CL4-BV-bound phages were recovered (1st screening). We repeated this screening process two more times (2nd screening and 3rd screening). If the number of CL4-bound phage is increased during the screening, the ratio of the incubated phage titers to the recovered phage titers will increase. As shown in Fig. 3A, the ratio was increased during screening from 4.5×10−7 to 5.5×10−5, indicating that the screening system for CL4 binders may work. Indeed, the number of monoclonal phage clones with high affinity to CL4-BV was increased after the 3rd screening compared with that after the 2nd screening (Fig. 3B).

Bottom Line: CL4-displaying BV interacted with a CL4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), but it did not interact with C-CPE that was mutated in its CL4-binding region.C-CPE did not interact with BV and CL1-displaying BV.We screened the library for CL4 binders by affinity to CL4-displaying BV, and we found that the novel CL4 binders modulated the tight-junction barrier.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bio-Functional Molecular Chemistry, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan.

ABSTRACT
Recent progress in cell biology has provided new insight into the claudin (CL) family of integral membrane proteins, which contains more than 20 members, as a target for pharmaceutical therapy. Few ligands for CL have been identified because it is difficult to prepare CL in an intact form. In the present study, we developed a method to screen for CL binders by using the budded baculovirus (BV) display system. CL4-displaying BV interacted with a CL4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), but it did not interact with C-CPE that was mutated in its CL4-binding region. C-CPE did not interact with BV and CL1-displaying BV. We used CL4-displaying BV to select CL4-binding phage in a mixture of a scFv-phage and C-CPE-phage. The percentage of C-CPE-phage in the phage mixture increased from 16.7% before selection to 92% after selection, indicating that CL-displaying BV may be useful for the selection of CL binders. We prepared a C-CPE phage library by mutating the functional amino acids. We screened the library for CL4 binders by affinity to CL4-displaying BV, and we found that the novel CL4 binders modulated the tight-junction barrier. These findings indicate that the CL-displaying BV system may be a promising method to produce a novel CL binder and modulator.

Show MeSH