Patent Application: US-53705009-A

Abstract:
we describe a polypeptide which binds and modulates the activity of a tumour suppressor polypeptide , for example p53 ; a nucleic acid molecule encoding said protein and screening methods which modulate the binding activity of said polypeptide for its target polypeptide .

Description:
cells were grown in culture in dulbecco &# 39 ; s modified eagle medium ( invitrogen ) supplemented with 10 % foetal calf serum . the cells used in this study were tera ( testicular tumour cell line ), rko ( colon carcinoma ), saos - 2 ( osteosarcoma ), h1299 ( lung carcinoma ), 293 ( embryonic kidney ), sk - mel - 37 ( melanoma ), mcf7 ( mammary epithelial ) and u2os ( osteosarcoma ). anti - v5 antibody was purchased from invitrogen . n - 20 cd20leu fitc - conjugated monoclonal antibody was from becton dickinson . transfections throughout were performed by calcium phosphate precipitation . the est containing the cdna encoding iaspp6c ( i . m . a . g . e . clone 4994121 ) was obtained from mrc geneservice ( cambridge , u . k .). the cdna was subcloned into pcdna3 . 1 / v5 - his - topo ( invitrogen ). pcdna3 . 1 iaspp , pcdna3 . 1 aspp2 , pcdna3 . 1 ce - iaspp and pcdna3 p53 have been described previously ( bergamaschi et al ., 2003 ; samuels - lev et al ., 2001 ). the iaspp6c truncations used in fig1 were generated by pcr - directed cloning into pcdna3 . 1 / v5 - his - topo . a modified pcdna3 vector that has had two v5 sequences inserted 5 ′ of the polylinker was used to generate n - terminally v5 - tagged iaspp6c . anti - iaspp6c antibodies pab18 ( rabbit polyclonal ) and sa4 . 1 ( mouse monoclonal ) were raised against the peptide rlqpalppeaqsvpelee ( amino acids 492 to 509 of iaspp6c ). anti iaspp6c mouse monoclonal antibody lx049 . 3 was raised against a c - terminal his - tagged fusion protein containing amino acids 459 to 639 of iaspp6c . the corresponding cdna was amplified by pcr and subcloned into pcrt7 / cttopo ( invitrogen ). the recombinant iaspp6c fragment was generated in bl21 star e . coli ( invitrogen ) by incubation with 1 mm iptg for 4 h followed by purification under denaturing conditions . cells were washed twice in pbs , then scraped into 1 ml pbs and pelleted at 400 g . the cells were lysed by incubating for 30 minutes at room temperature in 8m urea , 1m thiourea , 0 . 5 % chaps , 50 mm dtt and 24 mm spermine , followed by centrifugation at 20 000 g for 20 minutes at 16 ° c . 30 μg protein was used for analysis by sds - page and immunoblotting as described previously ( yap et al ., 2000 ). cells were lysed by incubating on ice in np40 lysis buffer ( 50 mm tris ph8 . 0 , 150 mm nacl , 1 mm edta , 1 % np40 and protease inhibitors ( complete protease inhibitor cocktail , roche )) for 45 minutes followed by centrifugation for 20 minutes at 20 00 g at 4 ° c . between 0 . 5 and 2 mg lysate was precleared by rotating for 1 h at 4 ° c . with protein g sepharose beads ( amersham biosciences ). following removal of the beads , the lysate was transferred to a fresh tube and rotated overnight with blocked protein g sepharose beads at 4 ° c . and approximately 1 μg of either a specific antibody or non - specific mouse or rabbit igg ( sigma ) as controls . the beads were then washed three times in ice cold np40 lysis buffer and the resulting complexes analysed by sds - page and immunoblot . oligonucleotides containing 19 bases of sequence present in both iaspp6c and iaspp cdnas were ligated into the psuper expression plasmid as described previously ( brummelkamp et al ., 2002 ). the plasmids were verified by sequencing . the complete sequences of the oligonucleotides used to generate the sirna are as follows with the cdna sequences shown in upper case : for transfection , 1 × 10 6 h1299 cells were plated into 10 cm dishes . cells were transfected with 3 μg of pmacs h - 2k k alongside either psuper or psuper - si - rna iaspp ( 10 μg ). 48 h after transfection , cells expressing the pmacs h - 2k k plasmid were separated using the macs system ( miltenyi biotec ) according to the manufacturer &# 39 ; s instructions . this gave rise to two populations of cells : h - 2k k expressing ( transfected ) cells and non - expressing ( non - transfected cells ). both cell populations were lysed with ripa buffer ( 150 mm nacl , 1 mm edta , 50 mm tris ph8 , 0 . 5 % deoxycholate , 1 % np40 , 0 . 1 % sds ) on ice for 30 minutes followed by centrifugation at 20 000 g for 30 minutes at 4 ° c . p53 and iaspp6c were translated in vitro with 35s - methionine using the tnt t7 quick coupled transcription / translation system ( promega ). the reticulocyte lysates containing each protein were combined as indicated and incubated together for 1 h at 30 ° c . lx049 . 3 antibody immobilised on protein g sepharose beads was added to the binding reactions and rotated at 4 ° c . for 16 h . the beads were then washed with pbs . the bound proteins were released in sds sample buffer and analysed by 10 % sds - page . results were visualised by autoradiography . the transcriptional assay was carried out as described previously ( samuels - lev et al ., 2001 ). flow cytometry 1 × 10 6 saos - 2 cells were plated in 10 cm dishes 24 - 48 h prior to transfection . all cells were transfected with 2 μg of pcmv cd20 as a transfection marker . the following plasmids were transfected as appropriate at the stated amounts : pcdna3 p53 ( 1 μg ), pcdna3 . 1 ce - iaspp ( 7 . 5 μg ), pcdna3 . 1 iaspp ( 7 . 5 μg ), pcdna3 . 1 iaspp6c ( 1 μg ), pcdna3 . 1 aspp2 ( 10 μg ). 2 μg iaspp6c truncations were used in fig1 . empty pcdna3 vector was used to equalise the total amount of dna in all samples . 36 h after transfection , both attached and floating cells were harvested and analysed as described previously ( hsieh et al ., 1997 ). saos - 2 . cells were seeded on cover slips in 24 well plates at 50 % density and transfected with 0 . 5 - 3 μg of plasmid encoding the iaspp6c truncations . 24 h after transfection the cells were fixed with 200 μl of 4 % paraformaldehyde in pbs for 12 minutes then permeabilised with 0 . 1 % triton - x100 in pbs for 4 minutes . expression of the iaspp6c constructs was detected using anti - v5 antibody ( 1 : 100 dilution in 0 . 2 % fish skin gelatin ) for 40 minutes followed by a tritc or fitc - conjugated secondary antibody for 20 minutes . bergamaschi , d ., samuels , y ., jin , b ., duraisingham , s ., crook , t . & amp ; 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