Hsp-antigen fusion and their use for immunization.
Immunization with antigenic peptide non-covalently associated with HSP elicits the peptide specific CD8+T cell response. The evidence encourages us to test the vaccination effect of recombinant HSP to which antigenic peptides are genetically fused. In the fusion protein, there should be no empty HSP molecules that failed to associate with the peptide of interest, like in vitro reconstitution method, therefore, promising effect may be easily obtained. Recombinant proteins expressed in Escherichia coli often form inclusion bodies and are thereby obtained as insoluble proteins or as proteins lacking their original functions. We describe here a simple and rapid refolding method of histidine-tagged recombinant hsp70/hsp70-peptide complex using a Ni(2+)-agarose column chromatography, without taking a process of dialysis to remove denaturants. The hsp70(hsp70-peptide complex) expressed in E. coli as a form of inclusion body was solubilized in 8 M urea containing buffer and applied to a Ni(2+)-agarose column. The bound hsp70 was refolded on the column by quick removal of urea with urea-free buffer and eluted with a denaturant-free and imidazole-containing buffer. The purified hsp70 was homogeneous and soluble. In addition, it had a very high ATPase activity and strong CTL inducing activity, whereas hsp70 prepared by conventional dialysis method had a negligible ATPase activity. This simple and rapid refolding method may provide a general method for a restoration of function (and/or immunization effect) and solubility of histidine-tagged recombinant HSP.
Udono
Heiichiro
H
Department of Molecular Medicine, Division of Immunology, Nagasaki University School of Medicine, 852-8523, Japan. udonoh@net.nagasaki-u.ac.jp
Saito
Takashi
T
Ogawa
Masayo
M
Yui
Yatsuyuki
Y
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000941:Antigens / Q000276:immunology; D002855:Chromatography, Thin Layer / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000276:immunology*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000276:immunology*; D007114:Immunization / Q000379:methods*; D007700:Kinetics; D010446:Peptide Fragments / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000276:immunology
D000941:Antigens; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D000251:Adenosine Triphosphatases