A streamlined, automated protocol for the production of milligram quantities of untagged recombinant rat lactate dehydrogenase A using ÄKTAxpress™

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Abstract

Abstract
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 ÄKTAxpress™ chromatography system. Cation exchange followed by size exclusion results in average final purity in excess of 93 % and yields ~ 14 milligrams per litre equivalent of original cell culture 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 Å. 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.
Original languageEnglish
Article numbere0146164
JournalPLoS ONE
Volume10
Issue number12
DOIs
Publication statusPublished - 30 Dec 2015

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