Browsing M.Sc. Biotechnology by Subject "Sec14"
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The Development of a Time-Resolved, Vesicle-Based, Fluorescence Assay for the Activity of the Lipid Kinase Pik1Pik1 is a yeast phosphatidylinositol-4 kinase that is critical for vesicular traffic to the plasma membrane and endosomes from the trans-Golgi (1, 2). Pik1 is a soluble enzyme, and the small myristoylated, Ca2+ binding, EF hand protein, Frequenin (Frq1) facilitates its membrane localization (3). It has been suggested that Pik1 finds its substrate, phosphatidylinositol (PI), within phospholipid bilayers with assistance from the PI/PC transfer protein Sec14 (4). It is proposed that Sec14 stimulates the activity of Pik1 by partially removing PI from the bilayer and presenting it to the kinase as a more suitable substrate (4). In order to test this hypothesis, the Bellbrook Labs Transcreener ADP2 FI Assay kit, a fluorescence-based enzyme assay kit that reports the production of ADP, was adapted to create a time-resolved, vesicle-based assay. The assay was developed and validated with the catalytic subunit of Protein Kinase A (PKA) and the commercial human PI kinase PIK3C3. An expression system for Pik1 was created in yeast. To preserve the stability of the 125 kDa enzyme it was co-expressed with both Frq1 and the heat shock protein, Cdc37, producing the Pik1-Frq1 complex with a removable 10xhistidine tag on Pik1. The growth of the yeast transformants was investigated using different culture conditions, media, and methods of inducing protein expression. The expression of the Pik1-Frq1 complex was confirmed on a Western blot that showed a band corresponding to the molecular weight of the complex. Next steps will involve optimizing the purification of the Pik1-Frq1 complex on a metal affinity resin, with the ultimate goal being to test the Sec14 presentation mechanism by measuring the kinase activity of Pik1 from purified fractions in the presence and absence of Sec14.