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Preliminary Expression and Purification Studies of the G-protein-coupled Estrogen Receptor (GPER) in Bacterial, Yeast, and Cell-free Systems

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Title: Preliminary Expression and Purification Studies of the G-protein-coupled Estrogen Receptor (GPER) in Bacterial, Yeast, and Cell-free Systems
Authors: Kurohara, Dane
Advisor: Ng, Ho Leung
Keywords: Estradiol
G-protein-coupled estrogen receptor
E. coli expression
S. cerevisiae expression
Cell-free protein synthesis
Issue Date: 26 Sep 2014
Publisher: University of Hawaii at Manoa
Abstract: Estrogens have important functions in almost every area of the body, where they regulate physiological processes present in both men and women. Fluctuations in estrogenic pathways, therefore, often lead to a variety of medical disorders including osteoporosis, neurodegenerative diseases, cardiovascular diseases, and several cancer strains (breast, endometrial, ovarian, and uterine cancers). Traditionally, estrogenic activity has been associated with the nuclear estrogen receptors ERα and ERβ, but within recent years a novel transmembrane receptor, called G-protein-coupled estrogen receptor (GPER; formerly known as GPR30), has been recognized to be associated with these hormonal pathways. Understanding the three-dimensional structure of proteins allows researchers to understand the intricate functions these molecules play in the body, as well as design drugs to modulate protein activities. In-depth structural analyses have already been performed on ERα and ERβ which have led to the development of ERα- and ERβ-selective drugs such as tamoxifen and raloxifene, yet to date, little is known about the atomic framework of GPER. Structural determination of proteins is often accomplished via x-ray crystallography, however, this process often requires large (milligram) amounts of isolated protein. To date, no method of GPER protein expression has been described, thus, the goal of this project was to provide preliminary expression characteristics of this membrane protein in three expression modalities: Escherichia coli, Saccharomyces cerevisiae, and cell-free systems. Both cellular methods are inexpensive and scalable systems that can be grown in an academic lab on the 1-10 L scale, however, isolation of membrane proteins in a solubilized, functional form involves a multi-step process during which a large portion of protein is often lost to proteolysis and aggregation. In contrast, cell-free expression systems utilize the translational machinery of E. coli cellular extracts to express a desired protein. This method of expression, although highly sensitive to reaction conditions, is a streamlined process permitting the highest degree of customization, where reagents and their concentrations can be artificially controlled and modified as desired. In this project, various constructs of GPER and a broad range of expression conditions were tested in each system to determine the most efficient method of protein production.
Pages/Duration: vi, 79 pages
URI/DOI: http://hdl.handle.net/10125/33661
Rights: All UHM Honors Projects are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Appears in Collections:Honors Projects for Biology



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