Sub-Cloning recombinant Drp1 into APEX2-GFP and overexpressing APEX2-GFP-Drp1 protein for potential mapping.

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Drp1 regulation and mechanism in mammalian fission machinery have been extensively studied in an attempt to have a better understanding of the fission machinery mechanism, human diseases such as neurodegenerative ones that are associated with its dysfunction, as well as understand how it plays an important role in the development of embryo. In this study, we have generated the recombinant Drp1 by insertion into pcDNA5/FRT/TO APEX2-GFP vector and verified it with restriction digestion and sequencing the insert. Even though, the presence of stop codon of EGFP was blocking the Drp1 expression which was corrected by single amino acid gene mutagenesis. From this experiment, we have enabled initial steps for further experiments to better understand the Drp1 interactome and with the help of APEX2-proximity labeling and green fluorescent protein, we can achieve a comprehensive map of Drp1 interactomes through mass spectrometry and analyze kinases and phosphatases that perform interactions with it.

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Drp1, cloning, mitochondrial fission
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