Improved purification and biologic activities of staphylococcal toxic shock syndrome toxin 1.
An improved method for producing highly purified toxic shock syndrome toxin 1 (TSST-1) by preparative isoelectric focusing in a Bio-Rad Rotofor cell and then chromatofocusing is described. Purification to homogeneity was confirmed by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 50 micrograms of protein was loaded), by immunoblotting with polyclonal rabbit antiserum raised against the crude culture supernatant used for purification, and by autoradiography after iodination and SDS-PAGE. Biologic activity was demonstrated by mitogenicity and cytokine induction (tumor necrosis factor alpha [TNF-alpha], interleukin 1-beta [IL-1 beta], and IL-6) of human peripheral blood mononuclear cells (PBMCs) and by lethality in New Zealand White rabbits following subcutaneous infusion. In contrast to commercial TSST-1 preparations, our TSST-1 preparation required the presence of both monocytes and T cells for the induction of TNF-alpha and IL-1 beta from human PBMCs. A 46-kDa contaminating protein in the commercial TSST-1 preparation, identified as staphylococcal lipase, was likely responsible for the induction of TNF-alpha and IL-1 beta from human monocytes in the absence of T cells, a biologic activity falsely attributed to purified TSST-1. Our improved purification procedure for TSST-1 provides a high yield and is both more rapid and less labor intensive than previously reported methods. Furthermore, our studies clearly demonstrate the need for stringent methods of purity assessment of TSST-1 preparations before ascribing to them their potent biologic activities.
Kum
W W
WW
Department of Medicine, University of British Columbia, Vancouver General Hospital, Canada.
Laupland
K B
KB
See
R H
RH
Chow
A W
AW
D000328:Adult; D000818:Animals; D001427:Bacterial Toxins; D002460:Cell Line; D004768:Enterotoxins / Q000302:isolation & purification* / Q000633:toxicity; D005260:Female; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis; D015850:Interleukin-6 / Q000096:biosynthesis; D011817:Rabbits; D013211:Staphylococcus aureus / Q000472:pathogenicity*; D018089:Superantigens; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D001427:Bacterial Toxins; D004768:Enterotoxins; D007375:Interleukin-1; D015850:Interleukin-6; D018089:Superantigens; D014409:Tumor Necrosis Factor-alpha; C030107:enterotoxin F, Staphylococcal