Elizabeth Petro - Bacterial expression strategies for several Sus scrofa diacylglycerol kinase alpha constructs: solubility challenges

Document created by Elizabeth Petro on Aug 22, 2014
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  Publication Details (including relevant citation   information):

    Elizabeth J. Petro and Daniel M. Raben. "Bacterial expression     strategies for several Sus scrofa diacylglycerol kinase alpha     constructs: solubility challenges." Scientific Reports    3, Article number: 1609. 5 April 2013.     doi:10.1038/srep01609  


  We pursued several strategies for expressing either full-length   Sus scrofa diacylglycerol kinase (DGK) alpha or the   catalytic domain (alphacat) in Escherichia coli.   Alphacat could be extracted, refolded, and purified from   inclusion bodies, but when subjected to analytical gel filtration   chromatography, it elutes in the void volume, in what we conclude   are microscopic aggregates unsuitable for x-ray crystallography.   Adding glutathione S-transferase, thioredoxin, or maltose binding   protein as N-terminal fusion tags did not improve alphacat's   solubility. Coexpressing with bacterial chaperones increased the   yield of alphacat in the supernatant after high-speed   centrifugation, but the protein still elutes in the void upon   analytical gel filtration chromatography. We believe our work   will be of interest to those interested in the structure of   eukaryotic DGKs, so that they know which expression strategies   have already been tried, as well as to those interested in   protein folding and those interested in chaperone/target-protein   interactions.

  Address (URL): http://www.nature.com/srep/2013/130405/srep01609/full/srep01609.html