Isolation of pertussis toxin subunit proteins by reverse-phase high-performance liquid chromatography and reconstitution of the holotoxin molecule.
Reverse-phase high-performance liquid chromatography on a column of trimethylsilylated silica gel (TSK-TMS 250) was utilized for the isolation of the subunit proteins of pertussis toxin (PT). Recovery up to 95% was obtained for each of the five distinct subunits with a high degree of homogeneity as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. None of the individual subunit proteins exhibited PT-related leukocytosis-promoting activity or the ability to bind haptoglobin; however, these activities were partially restored when an equimolar mixture of the isolated subunit in 6 M guanidine-HCl was diluted from this chaotropic agent. The complex macromolecule subsequently isolated from the mixture displayed subunit composition and biological activities indistinguishable from those of native PT, indicating that the toxin molecule had been reassembled.
Yamakawa
Y
Y
Department of Applied Immunology, National Institute of Health, Tokyo, Japan.
Sato
H
H
Sato
Y
Y
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006242:Haptoglobins / Q000378:metabolism; D007964:Leukocytosis / Q000139:chemically induced; D051379:Mice; D008970:Molecular Weight; D037342:Pertussis Toxin; D011489:Protein Denaturation; D010566:Virulence Factors, Bordetella / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology
D000596:Amino Acids; D006242:Haptoglobins; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin