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

• SAITO Akihiro

  National Institute of Agrobiological Sciences Department of Bioresources Chemistry, Faculty of Horticulture, Chiba University

• KAKU Hanae

  National Institute of Agrobiological Sciences

• MINAMI Eiichi

  National Institute of Agrobiological Sciences

• FUJII Takeshi

  National Institute of Agro-Environmental Sciences

• ANDO Akikazu

  Department of Bioresources Chemistry, Faculty of Horticulture, Chiba University

• NAGATA Yoshiho

  Department of Bioresources Chemistry, Faculty of Horticulture, Chiba University

• SCHREMPF Hildgund

  FB Biologie/Chemie, Universität Osnabrück

• MIYASHITA Kiyotaka

  National Institute of Agro-Environmental Sciences

Bibliographic Information

Other Title

• 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
• An Enzyme-Linked Immunosorbent Assay (ELISA) to Determine the Specificity of the Sugar-Binding Protein NgcE, a Component of the ABC Transporter for<i>N</i>-Acetylglucosamine in<i>Streptomyces olivaceoviridis</i>

Abstract

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.

Journal

• Bioscience, Biotechnology, and Biochemistry

  Bioscience, Biotechnology, and Biochemistry 70 (1), 237-242, 2006

  Japan Society for Bioscience, Biotechnology, and Agrochemistry

Keywords

• sugar-binding protein
• ATP-binding cassette (ABC) transporter
• neoglycoprotein
• chitinoligosaccharide
• <I>Streptomyces olivaceoviridis</I>

Details

• CRID: 1390282681452267392
• NII Article ID: 10018535629
• NII Book ID: AA10824164
• DOI: 10.1271/bbb.70.237
• ISSN: 13476947
• ISSN: 09168451
• NDL BIB ID: 7791735
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I7791735
• Web Site: http://www.tandfonline.com/doi/pdf/10.1271/bbb.70.237
• Text Lang: en
• Data Source:
  • JaLC
  • NDL
  • Crossref
  • CiNii Articles
• Abstract License Flag: Disallowed