Bacterial expression strategies for several Sus scrofa diacylglycerol kinase alpha constructs: solubility challenges.

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dc.contributor.authorPetro, Elizabeth J.
dc.contributor.authorRaben, Daniel M.
dc.date.accessioned2014-04-04T15:06:13Z
dc.date.available2014-04-04T15:06:13Z
dc.date.issued2013-04-05
dc.descriptionPMC3617429en_US
dc.description.abstractWe 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 interactionsen_US
dc.description.sponsorshipJH Libraries Open Access Funden_US
dc.identifier.citationdoi: 10.1038/srep01609.en_US
dc.identifier.issn2045-2322
dc.identifier.urihttp://jhir.library.jhu.edu/handle/1774.2/36742
dc.language.isoen_USen_US
dc.publisherNature Publishing Groupen_US
dc.relation.ispartofseriesScientific Reports;v. 3 p. 1609
dc.subjectSus scrofaen_US
dc.subjectProtein Engineeringen_US
dc.subjectGene Expression Regulation, Bacterialen_US
dc.subjectEscherichia colien_US
dc.subjectDiacylglycerol Kinaseen_US
dc.titleBacterial expression strategies for several Sus scrofa diacylglycerol kinase alpha constructs: solubility challenges.en_US
dc.typeArticleen_US
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