Protein Design, Expression, and Purification Core This proposal represents a unique academic effort that weds structural biology, molecular mycology, and vertebrate pathology to target the signal transduction pathways that allow pathogenic fungi to survive in humans. The structural approaches, which are essential to the success of the proposed Aims, will require the production of large quantities of pure and homogeneous proteins. Many of these proteins or protein complexes will be difficult to express and those that do express may also be difficult to produce in a soluble form. Thus, it is likely that multiple expression constructs will need to be tested for production of these soluble and pure target proteins.
The Specific Aims of this grant application are much better served if these activities are centralized as cost effectiveness is greatly enhanced by the removal of redundant efforts and the ability to bring to all projects technical scientists whose sole purpose is to maximize the output of biochemically active proteins. Thus, there are three general aims of goals of the Protein Design, Expression and Purification Core (PDEP):
Specific Aim 1 Generation of multiple expression constructs for target proteins and complexes. Genomic DNA, or artificially produced genes, will be inserted into multiple commercially available vectors providing a high likelihood of success.
Specific Aim 2 Testing for target constructs protein expression levels and solubility. Once cloned the PDEP will optimize the overexpression of soluble proteins using a 96 well plate format and multiple expression systems.
Specific Aim 3 Testing and optimizing expression constructs and performing final purification. The PDEP will purify proteins through affinity resins and help isotopically label proteins for NMR studies. The proteins will be distributed to core members for biochemical and structural studies. The most crucial goals of these studies will be high-resolution structure determination and the subsequent design of specific inhibitors.
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