Purification Of PRAS40 And 14-3-3, And Interaction Study Between Them By Yeast Two-hybrid System.

Grace Y.Park and her co-researchers use a combination of the 14-3-3 protein and anti-pAkt substrate antibodies to screen and isolate substrates of Akt. The major 14-3-3 binding protein observed in cells after insulin treatment was a 40-kDa molecule. This protein (designated PRAS40) was purified,sequenced, and identified as a proline-rich molecule without any major homology to other proteins in the database and also lacking any recognizable domains. They have demonstrated that this protein is a substrate of Akt by showing that it can be phosphorylated in vitro with purified Akt, that the activation of an inducible Akt (called mer-Akt) was alone sufficient to induce PRAS40 phosphorylation, and that the phosphorylation of this protein was decreased in cells lacking Akt1 and Akt2. PRAS40 expressed in all tissues and highly in liver and heart. Although the function of this protein is not known, the presence of several proline-rich, regions may allow it to interact with various SH3 or WW domain-containing proteins, thereby modifying the function of these molecules.The 14-3-3 protein family is highly conserved and is represented throughout the eukaryotic branch of life. The proteins were discovered in 1967 during a study of the soluble acidic proteins of the mammalian brain and named on the basis of fraction number during DEAE-cellulose chromatography and location after starch gel electrophoresis. The major native forms of 14-3-3s are homo-and hetero-dimers, the biological functions of which are to interact physically with specific client proteins and thereby effect a change in the client. As a result, 14-3-3s are involved in a vast array of processes such as the response to stress, cell-cycle control, and apoptosis, serving as adapters, activators, and repressors.Plasmid vector pDEST-17-PRAS40 and pDEST-15-14-3-3 are used to express and purify the GST-PRAS40 and HIS-14-3-3 reorganization fusion protein. The transfected E.Coli were cultured till the OD600 highly to 0.5, then induced by IPTG. SDS-PAGE used to detect the fusion proteins expression and it is show that the fusion proteins were succeed in inducing.PRAS40 and 14-3-3 were purified by Sepharose affinity column.To study the interaction between PRAS40 (Proline-rich-Akt Substate,40kDa) with 14-3-3 and to study the biological function of PRAS40 and 14-3-3, as to find new drug target, we construct expression vector pJG-PRAS40 (transcriptional activity plasmid) and pEG-PRAS40(DNA-binding plasmid) in yeast using gateway cloning technology, then the plasmid of pEG-PRAS40/pJG-PRAS40 is co-transformed into yeast cell with each pJG-14-3-3 /pEG-14-3-3 isoform plasmid. The co-transformants were tested byβ-galactosidase activity analysis and leucine medium growth analysis was used to study the interaction between PRAS40 and 14-3-3. The construction of pJG-PRAS40 and pEG-PRAS40 verified to be succeeded by elctrophoresis of enzyme digestion.With the system of yeast two-hybrid, PRAS40 was identified to interact with 14-3-3 isoforms: tau, beta, epsilon and zeta. Epsilon'ability of interaction to PRAS40 is stronger than beta and zeta'. Tau' ability is faintish.The role of PRAS40 interact with 14-3-3 is detected,PRAS40 and 14-3-3 fusion proteins are purified, this work can be useful in knowing the relations between PRAS40 and 14-3-3s and studying the biological function of PRAS40 and 14-3-3, as to finding new drug target.