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High Throughput Purification of Proteins for Biochemical and Structural Analysis

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tRNA Modifications in Yeast

Highly purified protein complexes isolated from yeast.

Recent work in this lab and the lab of E. Grayhack has focused on establishing yeast as the eukaryote of choice for high throughput purification of yeast and other eukaryotic proteins for biochemical analysis and structural biology. Although expression in E. coli has been used for biochemical analysis and for the determination of a large number of protein structures, expression of eukaryotic proteins in E. coli often results in limited solubility, as well as in the absence of post-translational modifications. To establish yeast as an organism for high throughput cloning, expression and purification, we have developed several methods.

We previously constructed the first genomic collection of yeast strains for parallel purification and biochemical analysis of yeast GST-ORF fusion proteins (in collaboration with Stan Fields at HHMI and the University of Washington), and used this collection for the rapid assignment of several biochemical activities to genes. Subsequently, we constructed a comprehensive library of yeast strains (the MORF collection, in collaboration with Mike Snyder at Yale and a group headed by Elizabeth Grayhack at Rochester), each strain of which expresses a yeast ORF (fused at its C-terminus to a tripartite tag) from a high copy plasmid under PGAL control, producing as much as 2 mg of purified protein per liter.

As part of the Center for High Throughput Structural Biology headed by George DeTitta at the Hauptman-Woodward Institute, we further developed the use of yeast for structural biology in two ways:

  1. E. Grayhack genetically modified yeast to permit high level incorporation of selenomethionine into proteins to facilitate obtaining phase information via multiwavelength anomalous dispersion. Previously, incorporation of selenomethionine into proteins has been difficult in yeast because of its toxicity.
  2. We developed a set of new vectors to permit facile cloning (by ligation independent cloning methods) of multiple tagged or untagged ORFs under inducible control.

Current research is directed at applying high throughput methods in E. coli and yeast to purify proteins involved in oxidative stress for structural analysis, in collaboration with a group headed by Eddie Snell at the Hauptman-Woodward Institute.

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