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A Streamlined, Automated Protocol for the Production of Milligram Quantities of Untagged Recombinant Rat Lactate Dehydrogenase A Using ÄKTAxpressTM

DOI: 10.1371/journal.pone.0146164 DOI Help
PMID: 26717415 PMID Help

Authors: Matthew Nowicki (University of Edinburgh) , Liz Blackburn (University of Edinburgh) , Iain Mcnae (University of Edinburgh) , Martin A. Wear (University of Edinburgh) , Jyotshna Kanungo
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Plos One , VOL 10

State: Published (Approved)
Published: December 2015
Diamond Proposal Number(s): 7613

Open Access Open Access

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 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 Å (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.

Subject Areas: Biology and Bio-materials

Instruments: I04-Macromolecular Crystallography