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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The ATP-binding cassette (ABC) transporter Ngc for <I>N</I>-acetylglucosamine (GlcNAc) of the chitin-degrader <I>Streptomyces olivaceoviridis</I> comprises the solute-binding protein NgcE, which has highest affinity for GlcNAc and <I>N</I>,<I>N</I>′-diacetylchitobiose {(GlcNAc)<SUB>2</SUB>} 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)<SUB>2</SUB> 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)<SUB>2</SUB>, which was fixed to wells in microtitre-plates. The NgcE protein was shown to bind efficiently to the immobilized BSA-APEA-(GlcNAc)<SUB>2</SUB>. In competition assays, the affinity of NgcE was 1,000-fold higher for GlcNAc and (GlcNAc)<SUB>2</SUB> than for (GlcNAc)<SUB>3</SUB> and (GlcNAc)<SUB>4</SUB>. 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.

収録刊行物

  • Bioscience, biotechnology, and biochemistry  

    Bioscience, biotechnology, and biochemistry 70(1), 237-242, 2006-01-23 

    Japan Society for Bioscience, Biotechnology, and Agrochemistry

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各種コード

  • NII論文ID(NAID)
    10018535629
  • NII書誌ID(NCID)
    AA10824164
  • 本文言語コード
    ENG
  • 資料種別
    ART
  • ISSN
    09168451
  • NDL 記事登録ID
    7791735
  • NDL 雑誌分類
    ZR7(科学技術--農林水産--農産) // ZR2(科学技術--生物学--生化学) // ZP1(科学技術--化学・化学工業)
  • NDL 請求記号
    Z53-G223
  • データ提供元
    CJP書誌  CJP引用  NDL  J-STAGE 
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