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    On developing a high-throughput gene-to-protein-to-structure pipeline and its application for the production of 10 crystal structures

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    Date
    2007-12
    Author
    Zhao, Min
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    Abstract
    By now, genome-sequencing projects have contributed to the systematical identification of a large number of proteins with unknown functions. A cost effective, high-throughput method for going from gene to structure, including cloning, protein production and crystallization, will help greatly to characterize these proteins. While a general high-throughput pipeline, which was required for the structural genomics program, was developing in our laboratory, it was soon realized that individual attention were needed in working with a large portion of the targeted protein samples. For example, some crystal-directed Òsalvaging and rescueÓ procedures were needed for those targets that would not yield satisfactory results by high-throughput methods. These procedures included additional purification, protein modification and stable domain isolation. After salvaging, 4 structures have been produced successfully out of 34 selected targets that had failed in the original high-throughput pipeline. A general salvaging strategy was then developed. In the next stage, these various rescue procedures were integrated into the general high-throughput gene-to-structure pipeline to enhance the capability of the pipeline. For an additional 35 targets, the improved pipeline produced 5 new structures. Our success rate was 14%, higher than the average success rate (7%) of Protein Structure Initiative centers by August 2004. The improved high-throughput gene-to-structure pipeline was also applied to a non-structural genomics but a functionally important target, the human p100 protein. The structure of the human p100 protein tudor domain helped to delineate the molecular basis of its function.
    URI
    http://purl.galileo.usg.edu/uga_etd/zhao_min_200712_phd
    http://hdl.handle.net/10724/24537
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    • University of Georgia Theses and Dissertations

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