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

• Published in: Bioscience, biotechnology, and biochemistry Vol. 70; no. 1; pp. 237 - 242
• Main Authors: SAITO, Akihiro, KAKU, Hanae, MINAMI, Eiichi, FUJII, Takeshi, ANDO, Akikazu, NAGATA, Yoshiho, SCHREMPF, Hildgund, MIYASHITA, Kiyotaka
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 01.01.2006; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: Acetylglucosamine - metabolism; Animals; ATP-binding cassette (ABC) transporter; ATP-Binding Cassette Transporters - metabolism; Biological and medical sciences; Cattle; chitinoligosaccharide; Enzyme-Linked Immunosorbent Assay; Fundamental and applied biological sciences. Psychology; Glycoproteins - metabolism; Molecular Structure; neoglycoprotein; Serum Albumin, Bovine - chemistry; Streptomyces - chemistry; Streptomyces - metabolism; Streptomyces olivaceoviridis; Substrate Specificity; sugar-binding protein; Surface Plasmon Resonance; Wheat Germ Agglutinins - metabolism; chitinoligosaccharide; sugar-binding protein; Streptomyces olivaceoviridis; Streptomycetaceae; ATP-binding cassette (ABC) transporter; Streptomyces; neoglycoprotein; Actinomycetales; Binding protein; Specificity; Bacteria; Actinomycetes; ATP; ELISA assay; ABC transporter
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.70.237

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.