Purification of multiple heat shock proteins from a single tumor sample.
Heat shock protein-based vaccines have been shown to immunize against cancer and infectious diseases in both prophylactic and therapeutic protocols. So far, four classes of heat shock proteins (HSPs) preparation: gp96, HSP90 (hsp86, hsp84), HSP70 (hsc70, hsp70) and calreticulin have been used successfully. The methods for purifying them individually are now readily available. However, since tumors are not always available in large quantity, a major challenge remains the development of a procedure to simultaneously isolate these HSPs from the same sample. We report here that hsp40, hsp60, hsc70, hsp70, hsp84, hsp86, and gp96 (grp94) but not BiP (grp78) and calreticulin can be separated from a single tumor sample in one step using heparin-agarose chromatography. Interestingly this procedure separates the HSP70 isoforms hsp70 from hsc70, but not the HSP90 isoforms hsp84 and hsp86. The three main immunogenic HSPs, gp96, hsp86/84, and hsc70 can be further isolated to homogeneity using additional purification methods. In addition, we have shown that the interaction of the chaperoned peptides with hsc70 and gp96 is not compromised during heparin chromatography. These observations provide a new method for preparation of multiple HSP-based vaccines, circumventing the sample size limitation, as well as providing the possibility to study how multiple HSPs can synergize in eliciting immunity.
Ménoret
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A
Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, CT 06030, USA. menoret@up.uchc.edu
Bell
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G
D000818:Animals; D000951:Antigens, Neoplasm / Q000302:isolation & purification; D019496:Cancer Vaccines / Q000276:immunology / Q000302:isolation & purification; D002352:Carrier Proteins / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D000091342:Endoplasmic Reticulum Chaperone BiP; D005354:Fibrosarcoma / Q000737:chemistry* / Q000276:immunology*; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins / Q000302:isolation & purification; D006360:Heat-Shock Proteins / Q000145:classification / Q000276:immunology* / Q000302:isolation & purification*; D051379:Mice; D008807:Mice, Inbred BALB C; D009363:Neoplasm Proteins / Q000276:immunology* / Q000302:isolation & purification*; D012685:Sepharose / Q000031:analogs & derivatives
D000951:Antigens, Neoplasm; D019496:Cancer Vaccines; D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; C494372:Hspa8 protein, mouse; D009363:Neoplasm Proteins; C037405:heparin-sepharose; C052646:sarcoma glycoprotein gp96 rejection antigens; D012685:Sepharose