5es3

Publication Abstract from PubMed

We developed an efficient, automated 2-step purification protocol for the production of milligram quantities of untagged recombinant rat lactate dehydrogenase A (rLDHA) from E. coli, using the AKTAxpress chromatography system. Cation exchange followed by size exclusion results in average final purity in excess of 93% and yields ~ 14 milligrams per 50 ml of original cell culture in EnPresso B media, in under 8 hrs, including all primary sample processing and column equilibration steps. The protein is highly active and coherent biophysically and a viable alternative to the more problematic human homolog for structural and ligand-binding studies; an apo structure of untagged rLDHA was solved to a resolution 2.29 A (PDB ID 5ES3). Our automated methodology uses generic commercially available pre-packed columns and simple buffers, and represents a robust standard method for the production of milligram amounts of untagged rLDHA, facilitating a novel fragment screening approach for new inhibitors.

A Streamlined, Automated Protocol for the Production of Milligram Quantities of Untagged Recombinant Rat Lactate Dehydrogenase A Using AKTAxpressTM.,Nowicki MW, Blackburn EA, McNae IW, Wear MA PLoS One. 2015 Dec 30;10(12):e0146164. doi: 10.1371/journal.pone.0146164., eCollection 2015. PMID:26717415^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.