Research Project
7130022
[unreadable] DESCRIPTION (provided by applicant): One of the major bottlenecks in determining the structures of pharmaceutically important, mammalian membrane proteins is obtaining sufficient pure, functional protein for sustained crystallization trials. Yeasts are commonly used for the heterologous production of soluble recombinant proteins. However, their potential as hosts for making membrane proteins has yet to be fully realized. Here, we intend to create a strain of yeast specifically tailored for higher eukaryotic membrane protein expression by genetically manipulating the yeast so that it makes the mammalian sterol, cholesterol, instead of the fungal one, ergosterol. Based on the ever-increasing body of evidence that shows that cholesterol is essential for the activity and stability of many mammalian membrane proteins, we suggest that yeast membranes containing cholesterol will be a superior environment for mammalian membrane protein production than those with ergosterol. We expect that the presence of cholesterol in yeast membranes will stabilize the recombinant membrane protein, and will result in increased production levels of the target protein compared to wild-type. We also expect that a wider range of proteins will be able to be made in the cholesterol biosynthetic yeast as compared to wild-type. To test our hypothesis, we intend to genetically modify a strain of S. cerivisiae so that it can make cholesterol. After each genetic manipulation, the sterol content of the engineered yeasts will be determined to confirm that the sterols that accumulate are as expected. Finally, the expression and activity of a range of human membrane proteins that have a known cholesterol-dependence (including selected G-protein coupled receptors and transporters) will be compared between the wild-type and cholesterol biosynthetic strains. By completing this, we will be able to assess whether or not a strain of S. cerevisiae that makes cholesterol is indeed a better host for mammalian membrane protein production than one that synthesizes ergosterol. 60% of all human therapeutics acts against G-protein coupled receptors and transporters. Despite this, not one of these targets has had its structure determined. This has completely prevented the creation of new drugs by design strategies based on the structure of the protein. The cholesterol biosynthetic yeast proposed here will provide an optimized environment for making milligram quantities of many mammalian membrane proteins, and will underpin sustained structural studies that will lead directly to structure-based drug design. [unreadable] [unreadable] [unreadable]
