A Streamlined, Automated Protocol for the Production of Milligram Quantities of Untagged Recombinant Rat Lactate Dehydrogenase A Using AKTAxpress (TM)

作者：Nowicki Matthew W; Blackburn Elizabeth A; McNae Iain W; Wear Martin A

来源：PLos One, 2015, 10(12): e0146164.

DOI：10.1371/journal.pone.0146164

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 (TM) chromatography system. Cation exchange followed by size exclusion results in average final purity in excess of 93% and yields similar to 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 angstrom (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.

• 出版日期2015-12-30