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.

Cannabichromene and Δ9-Tetrahydrocannabinolic Acid Identified as Lactate Dehydrogenase-A Inhibitors by in Silico and in Vitro Screening

“…The specificity loop is an unstructured segment that opens to admit substrate and cofactor to the active site and subsequently encloses them . To emulate the binding site rearrangement typical of inhibitor binding, an “open” conformation structure of human LDHA was used for docking . Docking of both CBC and Δ 9 -THCA to LDHA predicted a binding location at the active site, overlapping the NADH cofactor at the nicotinamide binding site (Figure ).…”

“…29 To emulate the binding site rearrangement typical of inhibitor binding, an "open" conformation structure of human LDHA was used for docking. 30 Docking of both CBC and Δ 9 -THCA to LDHA predicted a binding location at the active site, overlapping the NADH cofactor at the nicotinamide binding site (Figure 4). The docking cluster in this site was selected as it had the highest docking score for both ligands.…”

“…Docking was performed using the SwissDock online docking server. , The protein structure used was the “A” chain of the 5ES3 crystal structure . Ligands were selected using their IDs in the ZINC15 ligand database, i.e., ZINC5821119 for Δ 9 -THCA and ZINC1531363 for CBC.…”

The fed-batch principle for the molecular biology lab: controlled nutrient diets in ready-made media improve production of recombinant proteins in Escherichia coli

“…A further study applied E. coli OverExpress C41 BL21(DE3) cells with a reduced activity of the T7 RNA polymerase and a higher tolerance membrane and other toxic proteins. These cells were used to produce tetrameric rat lactate dehydrogenase in 50 ml EnPresso growth system at the same level as obtained with 1 L traditional LB medium [ 53 ]. The authors succeeded with the purified protein to obtain the crystal structure.…”

A multi-column plate adapter provides an economical and versatile high-throughput protein purification system

“…Protein purification will continue to be a key component in development of new therapeutics [34, 35]. Biophysical techniques in drug discovery still require milligram yields of pure protein, creating the need to improve expression and purification time and cost [1, 6, 36, 37].…”