title: Purification of Shiga-like toxin 1 by pigeon egg white glycoproteins immobilized on Sepharose gels.
abstract: The galabiose structure Galalpha1-4Gal is rarely found in natural glycoproteins, but is abundantly present in pigeon egg white proteins as Galalpha(1-4)Galbeta(1-4)GlcNAc termini. Pigeon ovalbumin, ovomucoid, or the whole egg white were immobilized on periodate-oxidized Sepharose CL-6B gels by reductive amination. These gels were found to bind Shiga-like toxin type 1 (SLT-1) specifically and efficiently. SLT-1 was eluted from the gel beads with 0.5 M melibiose, which was more efficient and milder than elution with 4.5 M MgCl(2). SLT-1 was purified to homogeneity from the crude extract of Escherichia coli SLT100 expressing SLT-1 by a single affinity chromatographic step in 83-88% yield. The capacity of the gel was estimated to be ca. 1mg toxin/ml gel. Interestingly, SLT-2 was not bound by these affinity gels containing Galalpha1-4Galbeta1-4GlcNAc termini. Since SLT-2 has been shown to bind to Galalpha1-4Galbeta1-4Glc-terminating compounds, our results suggest that Glc in globotriose moiety is important for binding SLT-2, and replacing the Glc with GlcNAc in this triose renders it ineffective for binding SLT-2.
authors.affiliation: Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tomoda
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Arai
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Koyama
authors.forename: Hidenori
authors.initials: H
authors.lastname: Matsui
authors.forename: Satoshi
authors.initials: S
authors.lastname: O mura
authors.forename: Rika
authors.initials: R
authors.lastname: Obata
authors.forename: Yuan C
authors.initials: YC
authors.lastname: Lee
mesh_terms: D000586:Amination; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D010856:Columbidae; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005782:Gels; D006023:Glycoproteins / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D009005:Monosaccharides / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry; D010059:Ovomucin / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D012685:Sepharose / Q000737:chemistry*; D022622:Shiga Toxin 1 / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity; D022641:Shiga Toxin 2 / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity
chemical_list: D005782:Gels; D006023:Glycoproteins; D009005:Monosaccharides; D009844:Oligosaccharides; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D010059:Ovomucin; D012685:Sepharose