title: 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.
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.
authors.affiliation: National Institute of Agrobiological Sciences, Ibaraki, Japan. takosaito@faculty.chiba-u.jp
authors.forename: Akihiro
authors.initials: A
authors.lastname: Saito
authors.forename: Hanae
authors.initials: H
authors.lastname: Kaku
authors.forename: Eiichi
authors.initials: E
authors.lastname: Minami
authors.forename: Takeshi
authors.initials: T
authors.lastname: Fujii
authors.forename: Akikazu
authors.initials: A
authors.lastname: Ando
authors.forename: Yoshiho
authors.initials: Y
authors.lastname: Nagata
authors.forename: Hildgund
authors.initials: H
authors.lastname: Schrempf
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Miyashita
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000117:Acetylglucosamine / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000378:metabolism; D015394:Molecular Structure; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013302:Streptomyces / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D014909:Wheat Germ Agglutinins / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D006023:Glycoproteins; D014909:Wheat Germ Agglutinins; D012710:Serum Albumin, Bovine; D000117:Acetylglucosamine