An Enzyme-Linked Immunosorbent Assay (ELISA) to Determine the Specificity of the Sugar-Binding Protein NgcE, a Component of the ABC Transporter for N-Acetylglucosamine in Streptomyces olivaceoviridis
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概要
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The ATP-binding cassette (ABC) transporter Ngc for N-acetylglucosamine (GlcNAc) of the chitin-degrader Streptomyces olivaceoviridis comprises the solute-binding protein NgcE, which has highest affinity for GlcNAc and N,N′-diacetylchitobiose {(GlcNAc)2} and reduced affinity for longer chitooligomers. NgcE was used to develop a generally applicable enzyme-linked immunosorbent assay (ELISA) system. As a prerequisite, the reducing end of (GlcNAc)2 was coupled with the ethylamino group of 2-(4-aminophenyl)ethylamine. The resulting conjugate was linked with amino groups of bovine serum albumin (BSA) to gain the neoglycoprotein BSA-APEA-(GlcNAc)2, which was fixed to wells in microtitre-plates. The NgcE protein was shown to bind efficiently to the immobilized BSA-APEA-(GlcNAc)2. In competition assays, the affinity of NgcE was 1,000-fold higher for GlcNAc and (GlcNAc)2 than for (GlcNAc)3 and (GlcNAc)4. These results are consistent with those previously obtained by surface plasmon resonance. Since the ELISA method can be performed very rapidly at low cost, it should be an efficient general tool to determine the affinity of a ligand to its cognate solute-binding protein.
- 2006-01-23
著者
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ANDO Akikazu
Department of Biotechnology, Graduate School of Science and Technology, Chiba University
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NAGATA Yoshiho
Department of Biotechnology, Graduate School of Science and Technology, Chiba University
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Fujii T
Kyoto Univ. Kyoto
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Fujii T
Department Of Applied Biological Chemistry The University Of Tokyo
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MINAMI Eiichi
National Institute of Agrobiological Sciences
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KAKU Hanae
National Institute of Agrobiological Sciences
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Saito Akiko
Biotechnology Center Toyama Prefecture
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Saito Akiko
Department Of Applied Biological Chemistry The University Of Tokyo
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Fujii Takeshi
National Institute for Agro-Environmental Sciences
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Fujii T
Inst. Biol. Sci. Univ. Tsukuba
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Schrempf Hildgund
Fb Biologie/chemie Universitat Osnabruck
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Ando A
Division Of Applied Life Sciences Graduate School Of Agriculture Kyoto University
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Fujii T
Div. Of Bioresource Sci. Graduate School Of Horticulture Chiba Univ.
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Nagata Y
Department Of Biotechnology University Of Tokyo
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Nagata Yuji
Department Of Applied And Environmental Chemistry Kitami Institute Of Technology
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Ando Akikazu
Department Of Bioresources Chemistry Faculty Of Horticulture Chiba University
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Ando Akikazu
Department Of Agricultural Chemistry Chiba University
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Nagata Yoshiho
Department Of Bioresources Chemistry Faculty Of Horticulture Chiba University
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Nagata Yoshiho
Dep. Of Bioresources Chemistry Fac. Of Horticulture Chiba Univ.
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SAITO Akihiro
National Institute for Agro-Environmental Sciences
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MIYASHITA Kiyotaka
National Institute for Agro-Environmental Sciences
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Miyashita K
National Institute For Agro-environmental Sciences
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Miyashita Kiyotaka
National Inst. For Agro-environmental Sciences
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Fujii Takeshi
National Inst. For Agro-environmental Sciences
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Ando Akikazu
Dep. Of Nanobiology Graduate School Of Advanced Integration Sci.
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Minami Eiichi
National Inst. Agrobiological Sci. Ibaraki Jpn
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Nagata Y
Department Of Bioresources Chemistry Faculty Of Horticulture Chiba University
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