Expression of Lactate Dehydrogenase (LDHA) WT and A320T Mutant

Abstract

The human genome has a length of approximately 3 billion base pairs, containing a total of over 20,000 genes, which can all be subjected to mutations that cause variations in the genome. Variations in a genome can be potentially either benign or pathogenic, but it can be difficult to distinguish whether a variant is one or the other, leading to tens of thousands being classified as variants of unknown significance (VUS). Our lab was particularly interested in the VUSs in lactate dehydrogenase (LDHA), an oxidoreductase enzyme that catalyzes the reversible conversion of pyruvate to lactate, an important component to anaerobic metabolism. Our lab is focusing on the A320T (alanine to threonine mutation at position 320) VUS, in which we cultured BL21 (DE3) and DH5α E. coli cell strains, so that we are able to clone wild type (WT) and A320T mutant enzymes. We eventually expressed and purified the two types of LDHA to observe the activity of each. Through expression and purification of both wild type and mutated forms of LDHA, we can observe the impact that the A320T-mutated VUS will have on the function of the LDHA protein, thus determining whether the VUS is benign or pathogenic to the enzyme function.