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Screening of Proteins Interacting with MOG1by Yeast Two-hybrid System

Author: JiLiYing
Tutor: KeTie
School: Huazhong University of Science and Technology
Course: Genetics
Keywords: Nav1.5 SCN5A MOG1 Protein Interaction Yeast Two-Hybrid System
CLC: Q343
Type: Master's thesis
Year: 2012
Downloads: 15
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The cardiac sodium channel α subunit Nav1.5is critical to the generation andpropagation of the action potential in the heart, and can cause lethal cardiac arrhythmiasand sudden death when mutated or deregulated. Protein-protein interactions are critical forthe normal function of Nav1.5. In2008, Wang and colleagues reported the identification ofa novel protein named MOG1interacted with Nav1.5by using the Yeast Two-HybridSystem and demonstrated that MOG1enhance Nav1.5activity and stimulate it’strafficking to the cell membrane. These results support a critical role of MOG1inregulating the cardiac sodium channel and indicated a new function of MOG1. Interstingly,using a candidate gene approach, one group recently identified novel mutation E83D inMOG1-encoding gene (gene symbol RANGRF) among patients with BrS, which showeda direct mechanism linking dysfucion of MOG1to cardiac arrhythmia.Due to its critical importance in cardiac physiology and human disease, studies todefine the regulatory proteins and other components of MOG1would be of great interest.We performed Yeast Two-Hybrid System to identify proteins that interact with MOG1.Firstly, the Human full length MOG1was amplified by PCR which used plasmidp3×FLAG-CMV-7.1-MOG1as template and cloned into vector pGBKT7to generate Baitplasmid pGBKT7-MOG1. Then we transformed pGBKT7-MOG1into yeast strainAH109to test the self-activation, we also transformed it into yeast strain Y187to examineits expression and toxicity. After that, we screened human fetal brain cDNA library, SDnutrient medium and α-gal were used to identify the positive clonies. We retested thephenotype of the148preliminary positive clonies, detected the α-gal activation and sortedby PCR to exclude the false positive ones. Plasmid DNA was isolated from each positiveclone and used to transform E. coli to prepare plasmids, which were directed sequenced byABI genetics analyzer. After sequence comparison and Blast analysis against the NCBIdatabase, two positive clones from the screening were found which encode the portion ofEPB42(Erythrocyte membrane protein band4.2) and BTBD6(BTB(POZ) domaincontaining6). In conclusion, we find two potential proteins that interacted with MOG1by usingYeast Two-Hybrid System. Further studies are needed to confirm the interaction betweenEPB42/BTBD6and MOG1, we also will continue to investigate the physiological roles ofthese specific interactions and potential pathophysiological mechanisms of the MOG1related cardiac channel pathy.

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