Novel peptide interacting with anti-apoptotic proteins of a bcl-2 family
An anti-apoptotic protein, bcl-2 technology, applied in the field of new peptides, can solve problems such as false negatives and false positives
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Embodiment 1
[0059] Example 1: Identification of the peptide described in SEQ.ID.NO.1
[0060] The conjugation protocol was utilized in yeast (Legrain et al., Nature Genetics, 1997, 16 , 277-282) were screened against three human cDNA libraries (placental, brain, cell line CEMC7) by the double-hybrid technique (Fields et al.).
[0061] 1) Preparation of "bait" and "capture" proteins
[0062] a) The "bait" used is:
[0063] - Bcl-XL(1-209) C-terminal truncation fused to the LexA DNA binding domain
[0064] - Bcl-2(1-211) C-terminal truncation fused to the LexA DNA binding domain
[0065] - Bcl-XL (1-209) C-terminal truncation fused to the LexA DNA binding domain
[0066] They were expressed in Saccharomyces cerevisiae (CG1945 or L40ΔGal4) and pre-cultured at 30°C in synthetic medium lacking tryptophan (DO-Trp) until DO 600nm Achieving between 0.1 and 0.5 inclusive. Dilute 50ml of the preculture (DO 600nm =0.006) and incubated overnight at 30°C.
[0067] b) Yeast pools containing...
Embodiment 2
[0080] Example 2: Comparison between the peptides described in Example 1 and Bcl-W and / or Bcl-XL and / or Bcl-2
[0081] Identification of interactions
[0082] 1) GST "settlement"
[0083] The peptide obtained from Example 1 and Interaction between Bcl-W and / or Bcl-XL and / or Bcl-2.
[0084] a) Synthesis of radiolabeled Bid
[0085] Labeled proteins were obtained using TNT quick master kit (Promega). Mix 40 μl of TNT mix with 2 μl (equivalent to 20 μCi) 35 S-methionine (Amersham), 1 μg of plasmid DNA encoding Bid and sufficient water to bring the volume to 50 μl were incubated at 30° C. for 90 minutes.
[0086] The number of fmoles / μl of radioactive protein produced was calculated based on the number of methionines in the protein.
[0087] b) GST "settlement"
[0088] Combine 4 fmole of radioactive Bid protein with 3 μg fusion protein glutathione-S-transferase-Bcl-XL (GST-Bcl-XL) or glutathione-S-transferase-Bcl-2 (GST-Bcl-2 ) or glutathione-S-transferase-Bcl-W...
Embodiment 3
[0098] Embodiment 3: can inhibit Bcl-W and / or Bcl-XL and / or Bcl-2 and obtain in embodiment 1 Screening Assays for Compounds Interacting Between Peptides
[0099]Compounds to be tested were dispensed into 384-well plates (Corning flat bottom) at a final concentration of 10 μg / ml. As a control, fill one well with an equal volume of buffer / solvent without the compound to be tested. The peptide obtained in Example 1 labeled with fluorescein was added to each well so as to give a final concentration of 15 nM. Then add the fusion protein GST-Bcl-XL, GST-Bcl-W or GST-Bcl-2, so that in the 2 HPO 4 A final concentration of 500 nM (Bcl-XL, Bcl-2) and 1 μM (Bcl-W) was obtained in a buffer of 20 mM pH 4, EDTA 1 mM, NaCl 50 mM and pluronic acid F-68 0.05%. Fluorescence polarization was then measured by an En indicator (Packard Perkin-Elmer). A significant decrease in the fluorescence polarization recorded in experiments performed with the test compound compared to that obtained wi...
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