Limits...
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.

Show MeSH

Related in: MedlinePlus

Selection of C-CPE-displaying phage by using the CL4-BV system.A) Interaction of C-CPE-displaying phage with CL4-BV. Wild-BV or CL4-BV was coated on an immunoplate, and then scFv-displaying phage or C-CPE-displaying phage was added to the BV-coated immunoplate at the indicated concentrations. The BV-bound phages were detected by ELISA with anti-M13 antibody as described in Materials and methods. Data are representative of two independent experiments. Data are means ± SD (n = 3). B) Enrichment of C-CPE-displaying phage by the BV system. A mixture of scFv-phage and C-CPE-phage (mixing ratio of scFv-phage to C-CPE-phage = 2∶10) was incubated with a CL4-BV-coated immunotube, and the bound phages were recovered. Each phage clone was identified by PCR amplification, followed by agarose gel electrophoresis. Upper and lower pictures are before and after the selection, respectively. The putative sizes of the PCR products are 856 and 523 bp in scFv and C-CPE, respectively. The data are representative of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3038848&req=5

pone-0016611-g002: Selection of C-CPE-displaying phage by using the CL4-BV system.A) Interaction of C-CPE-displaying phage with CL4-BV. Wild-BV or CL4-BV was coated on an immunoplate, and then scFv-displaying phage or C-CPE-displaying phage was added to the BV-coated immunoplate at the indicated concentrations. The BV-bound phages were detected by ELISA with anti-M13 antibody as described in Materials and methods. Data are representative of two independent experiments. Data are means ± SD (n = 3). B) Enrichment of C-CPE-displaying phage by the BV system. A mixture of scFv-phage and C-CPE-phage (mixing ratio of scFv-phage to C-CPE-phage = 2∶10) was incubated with a CL4-BV-coated immunotube, and the bound phages were recovered. Each phage clone was identified by PCR amplification, followed by agarose gel electrophoresis. Upper and lower pictures are before and after the selection, respectively. The putative sizes of the PCR products are 856 and 523 bp in scFv and C-CPE, respectively. The data are representative of two independent experiments.

Mentions: We next examined the interaction between C-CPE-phage and CL4-BV. As shown in Fig. 2A, C-CPE-phage bound to CL4-BV but not to wild-BV, and a scFv-phage did not bind to CL4-BV. To determine if CL-BV can be used to select CL binders, we prepared a mixture of C-CPE-phage and scFv-phage at a ratio of 2∶10 and used CL4-BV to select CL4-binding phage in the mixtures. The amount of C-CPE-phage was increased to 11 of 12 clones in the mixture (Fig. 2B), indicating that CL-BV may be useful in the preparation of CL binders.


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)

Selection of C-CPE-displaying phage by using the CL4-BV system.A) Interaction of C-CPE-displaying phage with CL4-BV. Wild-BV or CL4-BV was coated on an immunoplate, and then scFv-displaying phage or C-CPE-displaying phage was added to the BV-coated immunoplate at the indicated concentrations. The BV-bound phages were detected by ELISA with anti-M13 antibody as described in Materials and methods. Data are representative of two independent experiments. Data are means ± SD (n = 3). B) Enrichment of C-CPE-displaying phage by the BV system. A mixture of scFv-phage and C-CPE-phage (mixing ratio of scFv-phage to C-CPE-phage = 2∶10) was incubated with a CL4-BV-coated immunotube, and the bound phages were recovered. Each phage clone was identified by PCR amplification, followed by agarose gel electrophoresis. Upper and lower pictures are before and after the selection, respectively. The putative sizes of the PCR products are 856 and 523 bp in scFv and C-CPE, respectively. The data are representative of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3038848&req=5

pone-0016611-g002: Selection of C-CPE-displaying phage by using the CL4-BV system.A) Interaction of C-CPE-displaying phage with CL4-BV. Wild-BV or CL4-BV was coated on an immunoplate, and then scFv-displaying phage or C-CPE-displaying phage was added to the BV-coated immunoplate at the indicated concentrations. The BV-bound phages were detected by ELISA with anti-M13 antibody as described in Materials and methods. Data are representative of two independent experiments. Data are means ± SD (n = 3). B) Enrichment of C-CPE-displaying phage by the BV system. A mixture of scFv-phage and C-CPE-phage (mixing ratio of scFv-phage to C-CPE-phage = 2∶10) was incubated with a CL4-BV-coated immunotube, and the bound phages were recovered. Each phage clone was identified by PCR amplification, followed by agarose gel electrophoresis. Upper and lower pictures are before and after the selection, respectively. The putative sizes of the PCR products are 856 and 523 bp in scFv and C-CPE, respectively. The data are representative of two independent experiments.
Mentions: We next examined the interaction between C-CPE-phage and CL4-BV. As shown in Fig. 2A, C-CPE-phage bound to CL4-BV but not to wild-BV, and a scFv-phage did not bind to CL4-BV. To determine if CL-BV can be used to select CL binders, we prepared a mixture of C-CPE-phage and scFv-phage at a ratio of 2∶10 and used CL4-BV to select CL4-binding phage in the mixtures. The amount of C-CPE-phage was increased to 11 of 12 clones in the mixture (Fig. 2B), indicating that CL-BV may be useful in the preparation of CL binders.

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
Related in: MedlinePlus