Specific Aims
The production of large quantities of proteins and complexes is a requisite first step towards achieving our Program Project’s overall aim, which is to delineate fungal-specific structural differences in targets that control three essential cell signalling pathways. Work thus far has revealed that a major bottleneck is obtaining adequate target proteins and complexes in soluble form. However, our preliminary studies indicate that if enough expression constructs and systems are produced and analysed, high level expression of soluble protein can be achieved. Thus, these findings underscore the central importance of a Core devoted to Protein Design, Expression and Purification (PDEP). The first step in the PDEP Core is target construct design. This step is crucial in obtaining a soluble target. Once constructs have been designed that produce soluble targets, the next step in the Core is to discover conditions that are optimal for high level expression of target proteins or complexes. The final step is to define parameters for target purification, as even minute contaminants can hinder crystallization. However, even highly pure proteins may not crystallize. Hence, the generation of multiple soluble and active constructs is required to ensure the highest likelihood for successful target crystallization, underscoring the importance of a dedicated and automated PDEP Core. Once the Core has produced large quantities of pure protein, they will be handed to the Project team. Although the Program’s investigators have extensive experience in gene cloning and protein overexpression, the PDEP will increase cost effectiveness by providing devoted technical experts to maximize the output of the numerous constructs required to obtain soluble, highly expressed protein and ultimately, well diffracting crystals. All three Projects will be supported through the following Specific Aims:
Aim 1: Generation of multiple expression constructs for target proteins and complexes. A major impediment in obtaining sufficient quantities of fungal and other eukaryotic proteins for structural studies is achieving high levels of expression of soluble targets. Thus, a principal objective of the PDEP is the generation of multiple expression constructs. These constructs will use either cloned genes from genomic DNA or codonoptimized genes. The genes will then be inserted into several expression plasmids. Target construct design strategies will also include co-expression systems, nested truncations, and covalent fusion between interacting targets. Finally, the Core will create single and multi site-directed mutants to test the functionality of proteins. These structure-guided mutations will use cutting edge, high throughput mutagenesis methodologies.
Aim 2: Testing target constructs for protein expression levels and solubility. The majority of the target expression and purification will be performed using E. coli as an expression system as it has proven to be suitable for the Aims of Projects 2 and 3. However, baculovirus will be included as an expression system as it has proven to be successful in the generation of soluble calcineurin targets (Project 1) in addition to E. coli expressed fusion constructs. Once cloned, the constructs will be tested for overexpression and solubility using both a 96-well plate format and 1 mL cultures; typical variables will be tested, including concentration of the inducing reagent, time of induction, temperature, and strains. The outcome of the solubility and expression level tests will determine which constructs move on to protein purification assessment and which design strategies are discontinued. Discontinued strategies include those showing no expression and/or solubility. Strategies subjected to modification would include those showing unacceptable levels of expression but are soluble or are highly expressed but not soluble.
Aim 3: Testing and optimizing expression constructs and performing final purification. A critical Core activity is to deduce conditions for obtaining target proteins at >98% purity. The PDEP Core will first use the appropriate affinity resin to purify each affinity-tagged target. Once optimal protein expression and purification conditions are obtained, the Core will produce isotopically labelled proteins for NMR studies and selenomethionine-substituted proteins for crystallographic structure determination by MAD/SAD phasing.
Synergies with Other Projects and Cores
The primary objective of the PDEP Core is to design soluble and pure target proteins for successful structure determinations in each project. Thus, the PDEP Core is a central driver of the Program’s overall goals. The Core will interact with each Project to supply the proteins needed for its structural biology aims. The Projects will provide feedback to the Core to optimize target design and purification procedures. The PDEP Core will be utilized consistently by all Projects to produce target proteins because our inhibitor design strategy is iterative. For example, structures will first be used to optimally design inhibitors. Structures will then be determined of the protein bound to the newly designed inhibitors. This will allow the fine-tuning of inhibitor design to the target binding site and the development of new inhibitors. The most promising inhibitors will then be tested in animal models (Inhibitor Testing Core) and the outcomes monitored to provide information on which compounds have optimal antifungal activity in vivo.
Team
Maria Schumaker, PhD
Principal Investigator