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

Elizabeth J. Petro, Daniel M. Raben

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Article number1609
JournalScientific reports
Volume3
DOIs
StatePublished - Apr 5 2013

ASJC Scopus subject areas

  • General

Fingerprint

Dive into the research topics of 'Bacterial expression strategies for several Sus scrofa diacylglycerol kinase alpha constructs: Solubility challenges'. Together they form a unique fingerprint.

Cite this