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.
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.
Saito
Akihiro
A
National Institute of Agrobiological Sciences, Ibaraki, Japan. takosaito@faculty.chiba-u.jp
Kaku
Hanae
H
Minami
Eiichi
E
Fujii
Takeshi
T
Ando
Akikazu
A
Nagata
Yoshiho
Y
Schrempf
Hildgund
H
Miyashita
Kiyotaka
K
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
D018528:ATP-Binding Cassette Transporters; D006023:Glycoproteins; D014909:Wheat Germ Agglutinins; D012710:Serum Albumin, Bovine; D000117:Acetylglucosamine