Of ICE protease family, CPP32 (apopain or Yama) plays a central role in different apoptotic pathways. To study the molecules regulating CPP32, yeast two_hybrid system was used to identify proteins (peptides) that inte...Of ICE protease family, CPP32 (apopain or Yama) plays a central role in different apoptotic pathways. To study the molecules regulating CPP32, yeast two_hybrid system was used to identify proteins (peptides) that interact with CPP32. First, the CPP32 gene was cloned into plasmid vector pGBT9. The resulting recombinant plasmid was designated as pGBT9/CPP. The pGBT9 /CPP plasmid was transformed into the yeast strain HF7C, then the leukemia library was introduced. The transformation mixture was plated on medium lacking Trp, Leu and His in the initial screen. Colonies growing on the selection medium were further assayed for β galactosidase activity. Within 42 Trp +Leu +His + colonies only 5 turned blue in the presence of X_Gal. Plasmid DNA from 5 positive yeast colonies was prepared respectively and used to transform HB101 by electroporation. The transformation mixture was plated on medium lacking Leu to be selected for the library plasmid. Finally, only one library plasmid, designated as pY1, was determined to be truly positive by retransformation of pGBT9/CPP and the library plasmid into HF7C. The inserted cDNA of pY1 encodes a peptide of 15 amino acids, suggesting that it may be the domain interacting with CPP32.展开更多
文摘Of ICE protease family, CPP32 (apopain or Yama) plays a central role in different apoptotic pathways. To study the molecules regulating CPP32, yeast two_hybrid system was used to identify proteins (peptides) that interact with CPP32. First, the CPP32 gene was cloned into plasmid vector pGBT9. The resulting recombinant plasmid was designated as pGBT9/CPP. The pGBT9 /CPP plasmid was transformed into the yeast strain HF7C, then the leukemia library was introduced. The transformation mixture was plated on medium lacking Trp, Leu and His in the initial screen. Colonies growing on the selection medium were further assayed for β galactosidase activity. Within 42 Trp +Leu +His + colonies only 5 turned blue in the presence of X_Gal. Plasmid DNA from 5 positive yeast colonies was prepared respectively and used to transform HB101 by electroporation. The transformation mixture was plated on medium lacking Leu to be selected for the library plasmid. Finally, only one library plasmid, designated as pY1, was determined to be truly positive by retransformation of pGBT9/CPP and the library plasmid into HF7C. The inserted cDNA of pY1 encodes a peptide of 15 amino acids, suggesting that it may be the domain interacting with CPP32.
