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Journal Articles

Expression of the extracellular region of the human interleukin-4 receptor $$alpha$$ chain and interleukin-13 receptor $$alpha$$1 chain by a silkworm-baculovirus system

Honjo, Eijiro; Shoyama, Yoshinari; Tamada, Taro; Shigematsu, Hideki*; Hatanaka, Takaaki*; Kanaji, Sachiko*; Arima, Kazuhiko*; Ito, Yuji*; Izuhara, Kenji*; Kuroki, Ryota

Protein Expression and Purification, 60(1), p.25 - 30, 2008/07

 Times Cited Count:13 Percentile:61.55(Biochemical Research Methods)

The receptor binding to Interleukin (IL)-13 is composed of the IL-13 receptor $$alpha$$1 chain (IL-13R $$alpha$$1) and the IL-4 receptor $$alpha$$ chain (IL-4R $$alpha$$). In order to investigate the interaction of IL-13 with IL-13R $$alpha$$1 and IL-4R $$alpha$$, the DNA fragments coding the extracellular regions of human IL-13R $$alpha$$1 and the IL-4R $$alpha$$ were fused with mouse Fc and expressed by a silkworm-baculovirus system. The expressed receptors were successfully purified by affinity chromatography using protein A, and the Fc region was removed by thrombin digestion. Size exclusion chromatography and SPR analysis revealed that mixture of IL-13 and IL-13R$$alpha$$1 showed predominant affinity to IL-4R$$alpha$$, although neither detectable affinity of IL-13 nor IL-13R$$alpha$$1 was observed against IL-4R$$alpha$$. Combining these data with the moderate affinity of IL-13 to IL-13R$$alpha$$1, this indicates that IL-13 first binds to IL-13R$$alpha$$1 and recruits consequently to IL-4R.

Oral presentation

Inhibition mechanism of Interleukin-13 (IL-13) signal by extracellular region of IL-13 receptor$$alpha$$2 chain

Matsumoto, Fumiko; Hatanaka, Takaaki*; Tamada, Taro; Honjo, Eijiro*; Ota, Shoichiro*; Ito, Yuji*; Izuhara, Kenji*; Kuroki, Ryota

no journal, , 

Interleukin-13 (IL-13) is a key cytokine critical to the development of T-cell-mediated humoral immune responses which are associated with allergy and asthma. IL-13 possesses two types of receptor: the heterodimer, composed of IL-13R$$alpha$$1 and IL-4R$$alpha$$, transducing the IL-13 signals; and the IL-13R$$alpha$$2, it has high affinity rather than IL-13R$$alpha$$1 against IL-13, acting as a nonsignaling "decoy" receptor. In order to investigate the inhibition mechanism of IL-13 signal by IL-13R$$alpha$$2, extra cellular region of IL-13R$$alpha$$2 was expressed by silkworm-baculovirus expression system after fusing the DNA cording the extracellular region of human IL-13R$$alpha$$2 with the Fc derived from mouse IgG2a. The expressed fusion protein (IL-13R$$alpha$$2)2-Fc was purified by a protein A column followed by an ion exchange column. The affinities of the ligand complexes, IL-13/IL-13R$$alpha$$1 and IL-13/IL-13R$$alpha$$2 to (IL-4R$$alpha$$)2-Fc were investigated to explore the inactivation mechanism of the IL-13R$$alpha$$1 signal with IL-13R$$alpha$$2 by surface plasmon resonance analysis. IL-13/IL-13R$$alpha$$1 complex has an affinity (KD=1.20$$times$$10$$^{-7}$$ M) with (IL-4Ra)2-Fc, whereas the IL-13/IL-13R$$alpha$$2 complex had no affinity with (IL-4R$$alpha$$)2-Fc. This observation suggests that IL-13R$$alpha$$2 does not inactivate the IL-13R$$alpha$$1 through formation of a ternary complex with IL-13 and IL-4R$$alpha$$, but removes the IL-13 with its higher affinity to IL-13 without forming a ternary complex.

Oral presentation

Single-chained HIV-1 protease linked by a disulfide bridge

Adachi, Motoyasu; Arai, Shigeki; Matsumoto, Fumiko; Kuroki, Ryota; Hatanaka, Takaaki*; Ito, Yuji*; Hidaka, Koshi*; Tsuda, Yuko*; Kiso, Yoshiaki*

no journal, , 

HIV protease is known as a drug target protein. To compare interactions between HIVPR and inhibitors, single-chained HIVPR linked by a disulfide bond was designed as N98C mutant. Both WT N98C and A17 N98C mutants were expressed as inclusion body and refolded by dilution method. The yield of the purified enzymes was similar to that of WT. We will report the results of interactions between inhibitors and WT N98C and A17 N98C mutants by physicochemical and crystal structure analyses.

Oral presentation

Interaction between the thrombopoietin receptor extra cellular region and its specific ligand

Matsumoto, Fumiko; Hatanaka, Takaaki*; Adachi, Motoyasu; Shimizu, Rumi; Tamada, Taro; Ito, Yuji*; Kuroki, Ryota

no journal, , 

no abstracts in English

Oral presentation

X-ray structure analysis of the single-chain derivatives of HIV-1 protease in complex with inhibitor

Adachi, Motoyasu; Hatanaka, Takaaki*; Ito, Yuji*; Hidaka, Koshi*; Tsuda, Yuko*; Kiso, Yoshiaki*; Kuroki, Ryota

no journal, , 

HIV protease is known as a drug target protein. It is important to clear relationship between structural data and kinetic and physicochemical parameters for drug design. We prepared single-chained enzyme linked by two amino acids and cross-liked enzyme bridged by disulfide bond. The two single-chained enzymes were expressed as inclusion body and refolded by dilution method. The purified enzyme complexed with inhibitor KNI-272 was crystallized, and solved the crystal structures. The designed sc- and cl-HIV-PR will be useful for evaluate the affinity of newly designed inhibitors from kinetic and thermodynamic point of view. Finally, we also report the results of analysis in affinity of inhibitors by surface plasmon resonance.

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