Purification of recombinant and endogenous HSP70s.
Heat shock proteins (HSPs) are powerful immunogens against the antigenic peptides they chaperone. The antigenic peptides are MHC I-binding peptides and their elongated precursors derived from tumor antigens, viral antigens, minor histocompatibility antigens, or model antigens. HSP-peptide complexes can immunize against tumors and pathogen-infected cells. Remarkably, HSPs do not immunize after elution of the peptides they chaperone, demonstrating that HSPs are not immunogenic per se, whereas HSP-peptide complexes are. Additionally, HSPs activate professional antigen presenting cells (APC) through specific receptor(s) to stimulate secretion of pro-inflammatory cytokines, up-regulation of co-stimulatory molecules and activation of dendritic cells. The mechanistic exploration of the role of the HSPs on the innate and adaptive component of the immune system requires their isolation in large quantity. On one hand, isolation of naturally formed HSP-peptide complexes is key to study their specific immunogenicity. On the other hand, purification of HSPs free of endotoxin contamination is an absolute requirement for the analysis of their ability to activate APC in vitro. This chapter describes a convenient and fast method of purification of endogenous and recombinant HSP of 70 kDa (HSP70) that addresses these two considerations.
Ménoret
Antoine
A
Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, CT 06030, USA. menoret@up.uchc.edu
D000818:Animals; D000911:Antibodies, Monoclonal; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity / Q000379:methods; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003001:Cloning, Molecular; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000302:isolation & purification*; D007202:Indicators and Reagents; D008099:Liver / Q000737:chemistry; D051379:Mice; D011994:Recombinant Proteins / Q000302:isolation & purification; D013154:Spleen / Q000737:chemistry
D000911:Antibodies, Monoclonal; D018840:HSP70 Heat-Shock Proteins; D007202:Indicators and Reagents; D011994:Recombinant Proteins