A Novel Screening System for Claudin Binder Using Baculoviral Display

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

CL4-binding phages.

<p>The sequences of C-CPE mutant in the CL4-binding phages were analyzed.</p

Preparation of CL4-displaying BV.

<p>A) Immunoblot analysis. Wild-BV and CL4-BV (0.1 µg/lane) were subjected to SDS-PAGE, followed by immunoblot analysis with anti-CL4 antibody. The lysate of CL4-expressing L (CL4/L) cells was used as a positive control. B, C) Interaction of a CL4 binder with CL4-BV. Immunotubes were coated with the wild-BV or CL4-BV, and C-CPE (B) or mutated C-CPE (C) was added to the BV-coated immunotubes at the indicated concentration. C-CPE bound to the BV-coated tubes was detected by ELISA with an anti-his-tag antibody.</p

C-CPE phage library.

<p>Phage clones were randomly picked up from the C-CPE phage library, and the amino acids sequences of C-CPE mutant were analyzed.</p

Screening of a novel CL4 binder.

<p>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 (1^st input phage). The phages bound to CL4-BV were recovered (1^st output phage). The CL4-BV-binding phages were subjected to two additional cycles of the incubation and wash step, resulting in 2^nd, 3^rd 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 2^nd and 3^rd 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.</p

Selection of C-CPE-displaying phage by using the CL4-BV system.

<p>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.</p