Identification of novel binding partners for tuberous sclerosis complex 2 (TSC2) by yeast two-hybrid approach.
Ključne besede
Povzetek
OBJECTIVE
To identify novel tuberous sclerosis complex (TSC2) binding partners by yeast two-hybrid screening.
METHODS
The yeast two-hybrid system DupLEX-A developed by OriGene Technologies and Mouse embryo and HeLa cells cDNA libraries were used in this study. The "bait" constructs, containing full-length and truncated form of TSC2 were prepared. The expression of all constructs in yeast was confirmed by immunoblotting with specific anti-LexA antibodies. The suitability of generated constructs for screening was tested in autoactivation and nuclear translocation assays. Screening of mouse embryo and HeLa cDNA libraries with selected baits was carried out according to manufacturer's recommendations. Positive clones were selected using double selection procedure and further confirmed in mating assay. Isolated cDNA clones were identified by automated DNA sequencing and database searching.
RESULTS
Extensive screening of two cDNA libraries from mouse embryo and HeLa cells with TSC2 baits led to the isolation of 102 positives clones. The specificity of interaction between TSC2 and binding proteins of selected clones was confirmed by mating assay for 83 clones. Sequencing of these clones indicated that they encode already known and novel TSC2-binding partners.
CONCLUSIONS
The isolation of several known TSC2-binding partners, such as several isoforms of 14-3-3, demonstrates the validity of generated bait constructs and screening conditions. In addition, we have found a number of novel interactors, which encode cytoskeletal proteins and signaling molecules, such as Ser/Thr phosphatases.
