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Your search keyword '"Kumagai, Izumi"' showing total 366 results
Start Over Author "Kumagai, Izumi" Publication Type Academic Journals
366 results on '"Kumagai, Izumi"'

7. Construction of IgG–Fab2 bispecific antibody via intein-mediated protein trans-splicing reaction.

Author

Yamada, Risa, Nakahara, Ishin, Kumagai, Izumi, Asano, Ryutaro, Nakanishi, Takeshi, and Makabe, Koki

Subjects
BISPECIFIC antibodies, IMMUNOGLOBULINS, PROTEINS, CHEMICAL yield, CYTOTOXINS, T cells, PRODUCTION methods
Abstract

A bispecific antibody (bsAb) is a class of engineered antibody molecules that simultaneously binds to two different antigens by having two kinds of antigen-binding domains. One of the major obstacles for the bsAb production is the incorrect chain-pairing problem, wherein each heavy and light chain should form pairings with the correct counterpart's chains, but the structural similarity of the incorrect partners also forms the incorrect pairings. This study aimed to demonstrate a bsAb construction method using intein-mediated protein trans-splicing to create IgG–Fab2–type bsAbs, which is a modified antibody with a structure in which two additional Fabs are linked to the N-terminus of the heavy chain of an IgG molecule. The chain-paring problem between a heavy chain and a light chain is circumvented by separate expression and purification of the IgG part and the Fab part. We found that the deletion of a possible glycosylation residue improved the reaction yield and side-reaction cleavage in the protein ligation step. The resulting bsAb, IgG–Fab2 (Her2/CD3), demonstrated target binding activity and cytotoxicity mediated by activated T cells. These results indicate that the use of the protein ligation to produce the IgG–Fab2 type bsAb will expand the bsAb production method. [ABSTRACT FROM AUTHOR]

Published
2023
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20. Direct immobilization of gold-binding antibody fragments for immunosensor applications

Author

Ibii, Takahisa, Kaieda, Masaru, Hatakeyama, Satoru, Shiotsuka, Hidenori, Watanabe, Hideki, Umetsu, Mitsuo, Kumagai, Izumi, and Imamura, Takeshi

Subjects
Antibodies -- Chemical properties, Antibodies -- Composition, Viral antibodies -- Chemical properties, Viral antibodies -- Composition, Gold -- Chemical properties, Biosensors -- Chemical properties, Biosensors -- Composition, Biosensors -- Design and construction, Engineering design -- Methods, Chemistry
Abstract

A novel method that enables antibody fragments to be immobilized on a sensor substrate with a high binding capability using molecular recognition has been developed. Using genetic engineering, we fabricated bispecific recombinant antibody fragments, which consist of two kinds of antibody fragments: a gold antibody fragment and a target molecule antibody fragment. Surface plasmon resonance (SPR) analysis indicated that these gold-binding bispecific antibody fragments bind directly to the gold substrate with high affinity ([K.sub.D] ~ [10.sup.-9] M). About 70% of the bispecific antibody fragments immobilized on the gold substrate retained their target protein-binding efficiency. The Sips isotherm was used to assess the heterogeneity in antibody affinity for the bispecific antibody fragments. The results showed that the immobilized bispecific antibody fragments exhibited an increased homogeneity of affinity ([K.sub.D]) to target molecules when compared with monospecific antibody fragments immobilized by conventional methods. The use of bispecific antibody fragments to directly immobilize antibody fragments on a solid-phase substrate offers a useful platform for immunosensor applications. 10.1021/ac100557k

Published
2010

21. Electrochemical detection of epidermal growth factor receptors on a single living cell surface by scanning electrochemical microscopy

Author

Takahashi, Yasufumi, Miyamoto, Takeshi, Shiku, Hitoshi, Asano, Ryutaro, Yasukawa, Tomoyuki, Kumagai, Izumi, and Matsue, Tomokazu

Subjects
Electrochemistry -- Research, Cell physiology -- Research, Scanning microscopy -- Methods, Scanning microscopy -- Usage, Chemistry
Abstract

A membrane protein on the surface of a single living mammalian cell was imaged by scanning electrochemical microscopy (SECM). The epidermal growth factor receptor (EGFR) is one of the key membrane proteins associated with cancer. It elicits a wide range of cell-type-specific responses, leading to cell proliferation, differentiation, apoptosis, and migration. To estimate EGFR expression levels by SECM, EGFR was labeled with alkaline phosphatase (ALP) via an antibody. The oxidation current of PAP (p-aminophenol) produced by the ALP-catalyzed reaction was monitored to estimate the density of cell surface EGFR. EGFR measurement by SECM has three advantages. First, a single adhesion cell can be measured without peeling it from the culture dish; second, it is possible to optimize labeling antibody concentrations by using living cells because detection of faradaic current is suitable for quantitative estimation in situ; and third, SECM measurements afford information on the expression state at the cell membrane at the single-cell level. In this study, we optimized the concentration of labeling antibody for EGFR at the cell surface and confirmed distinct differences in EGFR expression levels among three types of cells. SECM measurements were compatible with the results of flow cytometry.

Published
2009

44. Human cystatin A is inactivated by engineered truncation. The NH2-terminal region of the cysteine proteinase inhibitor is essential for expression of its inhibitory activity

Author

Shibuya, Kazunori, Kaji, Hiroyuki, Itoh, Takehito, Ohyama, Yukihito, Tsujikami, Akiyoshi, Tate, Shin-ichi, Takeda, Atsushi, Kumagai, Izumi, Hirao, Ichiro, Miura, Kin-ichiro, Inagaki, Fuyuhiko, and Samejima, Tatsuya

Subjects
Enzyme inhibitors -- Research, Cysteine proteinases -- Research, Biological sciences, Chemistry
Abstract

A comparaison of the inhibitory activity of NH2-terminal human cysteine proteinase fused with porcine adenylate kinase shows that the amino-terminal is necessary for its activity. Removal of six residues from the amino-terminal decreases the inhibitory activity while removal of 15 residues completely inactivates the inhibitor. However the removal of the Met residue from the amino-terminal or substitution of the Pro3 with Leu residue does not affect the inhibitory activity. Substitution of the Gky75 with His residue decreases the loss of activity.

Published
1995

48. Functional conversion of the homologous proteins alpha-lactalbumin and lysozyme by exon exchange

Author

Kumagai, Izumi, Takeda, Shigeki, and Miura, Kin-Ichiro

Subjects
Lysozyme -- Research, Recombinant proteins -- Research, Enzymes, Phylogeny -- Research, Science and technology
Abstract

The gene organization of alpha-lactalbumin and c-type lysozyme are highly similar, although they exhibit highly different functions. The exon 2 region of goat alpha-lactalbumin was replaced with the corresponding region of hen lysozyme. The resulting hybrid protein exhibited lyoszyme-like catalytic activity. The results provide insights in the evolution of two structurally similar proteins with highly divergent functions.

Published
1992

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