A rapid, single-step purification method for immunogenic members of the hsp 70 family: validation and application.
Gelatin affinity chromatography has been developed as a simple one-step procedure for purification of members of the hsp 70 kDa family from MDBK cells (a bovine epithelioid cell line), rat liver microsomes and three different protozoan parasites. The ability of the isolated proteins to bind to denatured proteins like gelatin together with their apparent molecular masses, constitutive and inducible expression and their release from gelatin-agarose beads by ATP suggested that these proteins are molecular chaperones. The identity of a gelatin bound, ATP released, 78 kDa protein isolated from rat liver was confirmed by comparison of its NH2-terminal sequence with that of grp 78/BiP from rat. A 68 kDa protein isolated from Trypanosoma brucei brucei (T.b. brucei) and proteins of 68 and 69 kDa from Leishmania donovani (L. donovani) using gelatin affinity chromatography reacted in Western blot analysis with a monoclonal antibody, 7.10, specific for members of the 70 kDa heat shock protein family derived from a wide variety of species. A different monoclonal antibody, SPA-820, which also recognises members of the hsp 70 kDa family, bound to proteins isolated from Theileria parva Muguga transformed lymphoblastoid cell lines (TpM). The gelatin bound ATP released proteins of 72 kDa from T.b. brucei and of 65, 69 and 72 kDa from TpM were detected by recovery sera of the cattle infected with T.b. brucei and T. parva, respectively.
Nandan
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International Laboratory for Research on Animal Diseases, Nairobi, Kenya.
Daubenberger
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C
Mpimbaza
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Pearson
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TW
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D056890:Eukaryota / Q000276:immunology; D005780:Gelatin; D018840:HSP70 Heat-Shock Proteins / Q000276:immunology* / Q000302:isolation & purification*; D008862:Microsomes, Liver / Q000276:immunology; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000276:immunology; D015800:Protozoan Proteins / Q000302:isolation & purification; D051381:Rats; D015203:Reproducibility of Results
D018840:HSP70 Heat-Shock Proteins; D015800:Protozoan Proteins; D005780:Gelatin