@article{53d541bc0a854b3b8563ca79af4b7ff2,
title = "Bacterial expression strategies for several Sus scrofa diacylglycerol kinase alpha constructs: Solubility challenges",
abstract = "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.",
author = "Petro, {Elizabeth J.} and Raben, {Daniel M.}",
note = "Funding Information: We would like to thank all members of the Raben laboratory, as well as Professors Daniel Leahy, Mario Amzel, and Sandra Gabelli (Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine) for helpful discussions; Dr. Bryan Geisbrecht of Professor Daniel Leahy{\textquoteright}s laboratory for providing the pT71myc plasmid; Dr. Rishi Porecha for cloning alphacat into pT71myc; Ms. Michele Ostroski for cloning alphacat into pGEX-4T2; Mr. David Bolduc of Professor Philip Cole{\textquoteright}s laboratory (Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine) for providing the chaperone plasmids; Dr. Becky Tu-Sekine for transforming the chaperone plasmids into BL21(DE3); Dr. Nadine Samara of Professor Cynthia Wolberger{\textquoteright}s laboratory (Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine) for providing the pET32a plasmid; Ms. Tonya Gilbert of Professor Sean Taverna{\textquoteright}s laboratory (Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine) for providing the pET28-HisMBP-FLAGpp plasmid; Dr. Agedi Boto of Professor Mario Amzel{\textquoteright}s laboratory for support scaling up the expression of alphacat in pT71myc in E. coli; Ms. Hana Goldschmidt for performing the protein electrophoresis shown in Figure 2b; Professor Albert Mildvan (Biological Chemistry, The Johns Hopkins University School of Medicine) for providing the SephadexH G-200 resin; the laboratory of Professor Gerald Hart (Biological Chemistry, The Johns Hopkins University School of Medicine) for the use of their shaking incubator, centrifuges, probe sonicator, and spectrophotometer; the laboratory of Professor Jun Liu (Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine) for the use of their spectrophotometer; the laboratory of Professor Jeffry Corden (Molecular Biology & Genetics, The Johns Hopkins University School of Medicine) for the use of their OdysseyH Infrared Imaging System; the Department of Cell Biology (The Johns Hopkins University School of Medicine) for the use of the ultracentrifuge; the Department of Biological Chemistry (The Johns Hopkins University School of Medicine) for the use of the film developer; and Dr. Sabina Muend for help editing the manuscript. This work was supported by an American Heart Association Predoctoral Fellowship (to EP) and by Grant GM059251 from the National Institutes of Health (to DMR).",
year = "2013",
month = apr,
day = "5",
doi = "10.1038/srep01609",
language = "English (US)",
volume = "3",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",
}