Three-step purification of gp96 from human liver tumor tissues suitable for isolation of gp96-bound peptides.
Glycoprotein 96 (gp96), a member of heat shock protein (HSP) family, plays important roles in both innate immunity by itself and in T cell adaptive immunity in complex with antigenic-specific peptides. Using ammonium sulfate precipitation, ConA-Sepharose affinity chromatography and anion exchange chromatography, we have successfully purified gp96 from human liver tumor tissues. Subsequently, the gp96-associated peptides were successfully isolated from the gp96 preparation in quantities sufficient for micro-sequencing and identification of the peptides [Lancet 357 (2001) 528]. This three-step purification method might be used as a universal technique for the easy and reproducible isolation of antigenic heat shock protein gp96 from liver tissue or even other tissues of different sources. This will inevitably trigger the search for antigenic-specific peptides bound to gp96. Here, we report the method in detail.
Department of Molecular Virology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, PR China.
Song-Dong
SD
Meng
Jian
J
Song
Zihe
Z
Rao
Po
P
Tien
George F
GF
Gao
D000645:Ammonium Sulfate; D000951:Antigens, Neoplasm / Q000302:isolation & purification* / Q000378:metabolism*; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000737:chemistry* / Q000821:virology; D011232:Chemical Precipitation; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D018840:HSP70 Heat-Shock Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D006512:Hepatitis B Core Antigens / Q000302:isolation & purification / Q000378:metabolism; D006515:Hepatitis B virus / Q000276:immunology; D006801:Humans; D008113:Liver Neoplasms / Q000737:chemistry* / Q000821:virology; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism; D010455:Peptides / Q000302:isolation & purification* / Q000378:metabolism*; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein
D000951:Antigens, Neoplasm; D018840:HSP70 Heat-Shock Proteins; D006512:Hepatitis B Core Antigens; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D010455:Peptides; C018551:glucose-regulated proteins; C052646:sarcoma glycoprotein gp96 rejection antigens; D000645:Ammonium Sulfate