Purification of the 90 kDa heat shock protein (hsp90) and simultaneous purification of hsp70/hsc70, hsp90 and hsp96 from mammalian tissues and cells using thiophilic interaction chromatography.
Heat shock proteins (HSPs) hsp70/hsc70, hsp90 and hsp96 were separated from mammalian cells and tissues on a gel obtained by the reaction of beta-mercaptoethanol with divinyl sulfone-activated Sepharose CL-6B (thiophilic gel or T-gel). Hsp90 revealed a much higher affinity towards the T-gel than the other HSPs. One-step thiophilic interaction chromatography of proteins resulted in a more than 80% purity and 85% yield of hsp90. Based on this observation, a simple and efficient method for the purification of hsp90 and a procedure for the simultaneous purification of several HSPs (hsp70/hsc70, hsp90 and hsp96) using thiophilic interaction chromatography was developed. All the HSPs were recovered with a high yield and purity (90-99%). The results indicated that the thiophilic gel is a highly efficient affinity matrix for the purification of hsp90 and can be used in the protocols of purification of different HSPs from cells and tissues of various animal species.
Skarga
Yuri
Y
Laboratory of Cell Cultures and Cellular Engineering, Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino 142290, Russia. yyskarga@gmail.ru
Vrublevskaya
Veronika
V
Evdokimovskaya
Yulia
Y
Morenkov
Oleg
O
D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002477:Cells; D002846:Chromatography, Affinity / Q000379:methods*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008322:Mammals / Q000378:metabolism*; D008623:Mercaptoethanol / Q000737:chemistry; D008970:Molecular Weight; D009928:Organ Specificity; D012685:Sepharose / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013450:Sulfones / Q000737:chemistry
D000951:Antigens, Neoplasm; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D013438:Sulfhydryl Compounds; D013450:Sulfones; C052646:sarcoma glycoprotein gp96 rejection antigens; C009873:divinyl sulfone; D008623:Mercaptoethanol; D012685:Sepharose