The Protein Expression Core Facility will be located at Florida State University and the University of Alabama at Birmingham. The protein expression group will use the expertise gained as a Core Membrane Protein Expression Facility for the recent Mtb Membrane Protein Structural Genomics Program (GM64676) to generate protocols for the sufficient expression of membrane proteins for all 3 Projects of this new Program. The former Program had two expression facilities;one at FSU and one at the UVa. Together these facilities expressed 203 inner membrane proteins from 350 Mtb open reading frames. Outer membrane proteins will be expressed and functionally analyzed at the University of Alabama at Birmingham to utilize the expertise of Niederweis. Additional expertise in the recombinant expression of outer membrane proteins from Marassi and in inner membrane proteins from Marassi and Opella will complement the expression skills of the Core A Facility. This Facility will also procure antibodies for the Program.
Aim 1 : Cloning, expression, and purification of full length membrane proteins -All of the membrane proteins on the Initial Target List have been cloned and most over-expressed. New clones will be made as necessary to target the expression to particular'cell fractions. Protocols have been developed and will be refined as necessary to express the proteins in sufficient quantities for the research in Projects 1, 2 and 3. Purification protocols are also in hand and will be optimized for the specific targets, as necessary.
Aim 2. Cloning, expression, and purification of membrane protein domains and mutants - While expression of full-length proteins is the first priority, there is growing evidence that transmembrane domains and extra-membranous domains often function independently and therefore some protein structural and assay efforts could utilize protein domain constructs. In addition, mutagenesis will be performed by this Core to stabilize domains or to insert Cys residues in an otherwise Cys-less construct to enable the collection of paramagnetic relaxation effect restraints that will be important for the structural efforts.
Aim 3. Uniform isotopic labeling and amino acid specific labeling - are essential for Projects 2 and 3. Uniform 15N, 13C and 2H labeling will be required as well as amino acid specific labeling (labeling of one type of amino acid, such as valine) of as many amino acids as possible. Effective and economic methods for isotope labeling will be used and refined.
Aim 4. Initial characterization of the proteins - Gel electrophoresis, circular dichroism, light scattering, analytical ultracentrifugation, and size exclusion chromatography will be used to characterize the homogeneity and stability of the sample preparations, the secondary structure content, and the oligomeric state of the protein.
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