Loading...
Major
Chemistry
Research Abstract
A detailed biochemical study of biological processes often involves isolating a protein of interest. Isolation of integral membrane proteins is especially challenging since the numerous hydrophobic regions are not soluble in an aqueous solution. The most common purification method for membrane proteins involves the suspension of the protein in a synthetic detergent micelle. While the use of detergent micelles is relatively simple, this artificial structure does not completely mimic the native membrane environment. Thus, the findings from the studies using detergent-solubilized protein may not translate to the function of the protein when embedded in a membrane. / To overcome the limitation of detergents, a membrane bi-layer system, called nanodiscs, has been developed.Nanodiscs are comprised of phospholipids and a membrane scaffolding protein (MSP). Phospholipids, the main component of cellular membranes, are composed of 2 hydrophobic tails and a hydrophilic head group. MSP is an amphipathic protein, meaning that it possesses both hydrophobic and hydrophilic regions. Due to the tendency of hydrophobic substances to clump together, this allows for the hydrophobic portions of MSP to preferentially encircle and form contacts with the hydrophobic tails of the phospholipids in an aqueous solution. The polar heads of the phospholipids and the hydrophilic portions of MSP interact with water, thus allowing a nanodisc to mimic a natural membrane bi-layer. /The focus of the research presented here is to optimize the purification and preparation of MSP1-D1, which specifically generates nanodiscs of approximately 10nm in diameter. The protein was first overexpressed in E. coli, and purified via Fast Protein Liquid Chromatography (FPLC). Next, the His-tag was removed via proteolytic cleavage. Cleavage conditions were extensively tested and optimized for efficiency. Ultimately, we found that cleavage for 1.5 hours at room temperature with a molar ratio of 98:1 MSP1:cleavage enzyme was ideal. Subsequent purification using 15mM imidazole produced the highest yield of purified, cleaved MSP1-D1.
Faculty Mentor/Advisor
Dr. Janet G. Yang
Purification and Preparation of MSP1-D1 protein to construct Nanodiscs
A detailed biochemical study of biological processes often involves isolating a protein of interest. Isolation of integral membrane proteins is especially challenging since the numerous hydrophobic regions are not soluble in an aqueous solution. The most common purification method for membrane proteins involves the suspension of the protein in a synthetic detergent micelle. While the use of detergent micelles is relatively simple, this artificial structure does not completely mimic the native membrane environment. Thus, the findings from the studies using detergent-solubilized protein may not translate to the function of the protein when embedded in a membrane. / To overcome the limitation of detergents, a membrane bi-layer system, called nanodiscs, has been developed.Nanodiscs are comprised of phospholipids and a membrane scaffolding protein (MSP). Phospholipids, the main component of cellular membranes, are composed of 2 hydrophobic tails and a hydrophilic head group. MSP is an amphipathic protein, meaning that it possesses both hydrophobic and hydrophilic regions. Due to the tendency of hydrophobic substances to clump together, this allows for the hydrophobic portions of MSP to preferentially encircle and form contacts with the hydrophobic tails of the phospholipids in an aqueous solution. The polar heads of the phospholipids and the hydrophilic portions of MSP interact with water, thus allowing a nanodisc to mimic a natural membrane bi-layer. /The focus of the research presented here is to optimize the purification and preparation of MSP1-D1, which specifically generates nanodiscs of approximately 10nm in diameter. The protein was first overexpressed in E. coli, and purified via Fast Protein Liquid Chromatography (FPLC). Next, the His-tag was removed via proteolytic cleavage. Cleavage conditions were extensively tested and optimized for efficiency. Ultimately, we found that cleavage for 1.5 hours at room temperature with a molar ratio of 98:1 MSP1:cleavage enzyme was ideal. Subsequent purification using 15mM imidazole produced the highest yield of purified, cleaved MSP1-D1.
