Reconstitution of active diphtheria toxin based on a hexahistidine tagged version of the B-fragment produced to high yields in bacteria.
Diphtheria toxin consists of an A-fragment that inactivates elongation factor 2 and a B-fragment that both binds to the toxin receptor and mediates translocation of the A-fragment across cellular membranes to the cytosol. Several fragments of the toxin and an inactive version of the holotoxin have been expressed in Escherichia coli, but the B-fragment alone has proven difficult to express. Only low levels of expression have been achieved. We have designed a hexahistidine tagged version of a modified diphtheria toxin B-fragment (DT-BHis) that can be expressed to high levels in E. coli. DT-BHis contains the entire diphtheria toxin B-fragment preceded by an alanine and succeeded by a leucine, a glutamic acid and a hexahistidine tag and could be purified in a single step using nickel-coated agarose beads to 85% homogeneity. DT-BHis bound specifically to the diphtheria toxin receptor and was able to compete out the effect of the wild type diphtheria toxin. Furthermore, DT-BHis was able to form pores in cellular membranes in a manner similar to the wild type B-fragment. The high yield makes DT-BHis a suitable tool in studies of diphtheria toxin interaction with cells or liposomes since functional diphtheria toxin was easily formed upon addition of A-fragment. The reconstituted diphtheria toxin showed toxicity in the same range as the wild type.
Spilsberg
Bjørn
B
Department of Biochemistry, Institute for Cancer Research, Faculty Division The Norwegian Radium Hospital, University of Oslo, Montebello, N-0310 Oslo, Norway.
Sandvig
Kirsten
K
Wälchli
Sébastien
S
D000818:Animals; D019149:Bioreactors; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017931:DNA Primers; D004167:Diphtheria Toxin / Q000302:isolation & purification* / Q000378:metabolism* / Q000633:toxicity; D004926:Escherichia coli; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D009842:Oligopeptides / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity; D018675:Toxicity Tests; D014709:Vero Cells
D017931:DNA Primers; D004167:Diphtheria Toxin; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D010446:Peptide Fragments; C029795:diphtheria toxin fragment A; C039171:diphtheria toxin fragment B; D006639:Histidine