Chemical Strategies to Enrich Malarial Proteins from Wheat Lysate

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Chemical Strategies to Enrich Malarial Proteins from Wheat Lysate

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Title: Chemical Strategies to Enrich Malarial Proteins from Wheat Lysate
Author: Maki, Jennifer
Abstract: Malaria is a major public health burden and developing the next generation of anti-malarials is vital to control the spread of disease. Protein arrays, which can investigate binding of many proteins to various probes simultaneously, are becoming an important tool in the drug development process. Results from protein arrays can be affected by the sample purity, as high amounts of protein from the translation system can mask positive interactions on the arrays. The results would be improved by separating the malarial protein from the translation system proteins. However, purifying the hundreds of proteins for arrays using traditional affinity fusion tags is extremely time-consuming. To reduce the time required to produce protein samples at the desired purity, enrichment schemes were developed that covalently attach a small molecule to the proteins in the wheat cell-free expression system. After translation, separation matrices with high specificity for the small molecule modification were added to remove the modified wheat proteins, leaving the malarial protein as a higher percentage in the sample (labeled an enriched sample). Three enrichment schemes, based on the affinities between biotin and streptavidin and histidine peptides and Co2+ ions, removed more than 675 µg (more than 85%) of the wheat protein. This resulted in samples with equal concentrations of malarial protein and wheat protein (approximately 0.5 mg/mL), down from the initial 20-fold excess of wheat proteins. Enrichment increased specific activity of two expressed enzymes, dihydrofolate reductase-thymidylate synthase and topoisomerase II, by 3.2- to 4.9-fold. Model protein arrays were created by immobilizing enriched protein samples and various controls. The enriched samples produced fluorescence signals 2.9- to 4.1-fold higher than the non-enriched controls. These enrichment schemes will decrease the time required for preparation of hundreds of protein samples to weeks instead of the months or years it would take with traditional affinity tags. Additionally, the increased fluorescence intensities seen on the arrays from enriched samples will provide better identification of binding interactions, especially for proteins with low expression yields or for weak binding interactions.
Description: Thesis (Ph.D.)--University of Washington, 2012
URI: http://hdl.handle.net/1773/20610
Author requested restriction: Restrict to UW for 1 year -- then make Open Access

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