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-ste p 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 prote ins to bind to denatured proteins like gelatin together with their app arent molecular masses, constitutive and inducible expression and thei r release from gelatin-agarose beads by ATP suggested that these prote ins are molecular chaperones. The identity of a gelatin bound, ATP rel eased, 78 kDa protein isolated from rat liver was confirmed by compari son of its NH2-terminal sequence with that of grp 78/BiP from rat. A 6 8 kDa protein isolated from Trypanosoma brucei brucei (T.b. brucei) an d proteins of 68 and 69 kDa from Leishmania donovani (L. donovani) usi ng gelatin affinity chromatography reacted in Western blot analysis wi th a monoclonal antibody, 7.10, specific for members of the 70 kDa hea t shock protein family derived from a wide variety of species. A diffe rent monoclonal antibody, SPA-820, which also recognises members of th e hsp 70 kDa family, bound to proteins isolated from Theileria parva M uguga transformed lymphoblastoid cell lines (TpM). The gelatin bound A TP released proteins of 72 kDa from T.b. brucei and of 65, 69 and 72 k Da from TpM were detected by recovery sera of the cattle infected with T.b. brucei and T. parva, respectively.