Peptide Expression and Purification to Elucidate the Mechanism of Vascular Alpha2C-Adrenoceptor Traslocation
Loading...
Date
2012-06
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
The Ohio State University
Abstract
In response to physiological cold temperatures, cutaneous blood vessels constrict to reduce heat loss. This constriction is mediated by smooth muscle G protein-coupled α2C-adrenoceptors (α2C-ARs), which translocate from the trans-Golgi to the plasma membrane upon cooling. The cellular mechanisms involved in receptor translocation are not fully understood; however, this translocation is hypothesized to be dependent upon a protein-protein interaction between the α2C-AR C-terminus and the protein filamin-2. This portion of the receptor translocation mechanism can be tested experimentally by decoy peptide interference.
The decoy peptide contains a TAT domain and an identical C-terminus to the α2C-AR, allowing the peptide to penetrate the cell membrane and mimic the α2C-AR, respectively. We predict that interfering with this interaction will inhibit translocation of α2C-ARs and subsequent receptor function. Therefore, the goal of this study is to generate and purify a decoy peptide, confirm delivery to human VSM cells, and assess cellular activity for endogenous α2C-ARs.
The DNA sequence encoding 11 amino acids of the cell-permeable HIV transactivator of transduction (TAT) domain was fused in frame with the α2C-AR C-terminus to generate the p-TAT-α2C-AR-C-terminus bacterial expression vector. BL21-CodonPlus competent cells were used to optimize peptide expression at 30°C. The parameters tested during bacterial growth included the presence of antibiotics, induction time, and growth temperature. Additionally, cell lysis, sonication and salt precipitation conditions were optimized. Peptide expression was confirmed by Western blot and Coomassie techniques. The peptide was purified by ammonium sulfate, ion-exchange chromatography and nickel column techniques. The peptide was successfully delivered to human VSM and HEK-293 cells, confirmed by microscopic visualization and western blot techniques, which will be utilized to assess activity of endogenous α2C-ARs. Additionally, these studies have broader implications for understanding the role of actin in protein translocation and cell surface delivery.
Description
Keywords
Peptide Expression and Purification, Biochemistry, Ion Exchange Chromatography, Cellular Mechanism