title: Expression and purification of Shiga-like toxin II B subunits.
abstract: Shiga-like toxins (SLTs), which are produced by certain strains of Escherichia coli, are composed of enzymatically active A and B subunit multimers responsible for the toxin's binding. We have previously purified large amounts of the SLT-I B subunit by using a hyperexpression vector in Vibrio cholerae under the control of the trc promoter. In this study we examined various expression vectors to maximize yields of the SLT-II B subunit. The SLT-II B subunit has been expressed by using both the T7 promoter and the tac promoter in E. coli. When expressed from a plasmid containing the structural gene for SLT-II B deleted of the leader sequence, SLT-II B was able to form multimers when cross-linked, although SLT-II B production from this plasmid was unreproducible. SLT-II B expressed in all three systems appeared to form unstable multimers, which did not readily bind to a monoclonal antibody which preferentially recognizes B subunit multimers. SLT-II B expression was not increased by moving any of the plasmids into V. cholerae. Polyclonal antibodies raised to SLT-II B in rabbits recognized B subunit in SLT-II holotoxin yet were poorly neutralizing. SLT-II B was also expressed as a fusion protein with maltose-binding protein and could be cleaved from maltose-binding protein with factor Xa. Although the expression vectors were able to make large amounts of SLT-II B, as determined by Western blotting (immunoblotting), the levels of purified SLT-II B subunit were low compared with those obtained previously for SLT-I B subunit, probably because of instability of the multimeric SLT-II B subunit.
authors: Division of Geographic Medicine and Infectious Diseases, New England Medical Center, Boston, Massachusetts 02111.
authors: D W
authors: DW
authors: Acheson
authors: S A
authors: SA
authors: De Breucker
authors: M
authors: M
authors: Jacewicz
authors: L L
authors: LL
authors: Lincicome
authors: A
authors: A
authors: Donohue-Rolfe
authors: A V
authors: AV
authors: Kane
authors: G T
authors: GT
authors: Keusch
mesh_terms: D018528:ATP-Binding Cassette Transporters; D000907:Antibodies, Bacterial; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D001483:Base Sequence; D001681:Biological Assay; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D022641:Shiga Toxin 2; D014734:Vibrio cholerae / Q000235:genetics
chemical_list: D018528:ATP-Binding Cassette Transporters; D000907:Antibodies, Bacterial; D001427:Bacterial Toxins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D022641:Shiga Toxin 2; C461599:maltose transport system, E coli