title: A Streamlined, Automated Protocol for the Production of Milligram Quantities of Untagged Recombinant Rat Lactate Dehydrogenase A Using ÄKTAxpressTM.
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.
authors.affiliation: The Edinburgh Protein Production Facility (EPPF), Wellcome Trust Centre for Cell Biology (WTCCB), University of Edinburgh, King's Buildings, Max Born Crescent, Mayfield Road, Edinburgh, United Kingdom.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Nowicki
authors.affiliation: The Edinburgh Protein Production Facility (EPPF), Wellcome Trust Centre for Cell Biology (WTCCB), University of Edinburgh, King's Buildings, Max Born Crescent, Mayfield Road, Edinburgh, United Kingdom.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Blackburn
authors.affiliation: The Edinburgh Protein Production Facility (EPPF), Wellcome Trust Centre for Cell Biology (WTCCB), University of Edinburgh, King's Buildings, Max Born Crescent, Mayfield Road, Edinburgh, United Kingdom.
authors.forename: Iain W
authors.initials: IW
authors.lastname: McNae
authors.affiliation: The Edinburgh Protein Production Facility (EPPF), Wellcome Trust Centre for Cell Biology (WTCCB), University of Edinburgh, King's Buildings, Max Born Crescent, Mayfield Road, Edinburgh, United Kingdom.
authors.forename: Martin A
authors.initials: MA
authors.lastname: Wear
mesh_terms: D000818:Animals; D057205:Automation, Laboratory / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods; D003470:Culture Media; D004926:Escherichia coli / Q000378:metabolism; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification; D007770:L-Lactate Dehydrogenase / Q000096:biosynthesis* / Q000302:isolation & purification; D000080864:Lactate Dehydrogenase 5; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D020349:Surface Plasmon Resonance / Q000379:methods
chemical_list: D003470:Culture Media; D007527:Isoenzymes; D011994:Recombinant Proteins; D007770:L-Lactate Dehydrogenase; D000080864:Lactate Dehydrogenase 5