Format

Send to

Choose Destination
Sci Rep. 2013;3:1609. doi: 10.1038/srep01609.

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

Author information

1
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.

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.

PMID:
23558375
PMCID:
PMC3617429
DOI:
10.1038/srep01609
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Nature Publishing Group Icon for PubMed Central
Loading ...
Support Center