Construction of the hemagglutinin (HA) epitope tags for Subunit H of Complex I in Escherichia coli

Complex I is a member of the respiratory chain in both bacteria and human mitochondria. Studies have shown that many human mitochondria disorders are related to the poor assembly of Complex I, rather than simple enzymatic malfunction [1,2,3,4]. For the purpose of analyzing the assembly of the thirteen subunits (A-N) found in the Escherichia coli homolog of the Complex I enzyme, antibodies are needed to recognize the individual subunits. Since the H subunit is the only subunit without an antibody, high-affinity antibodies are needed to be produced from the HA epitope tags. The tags were introduced separately by the polymerase chain reaction (PCR) method in two different plasmids, one containing genes G-H, and the other containing H-I-J-K. DNA sequencing indicated high confidence in HA complexes. After growth of cell cultures, an induced expression of these genes by arabinose verified expression of the protein with induced tag. Then, a western blot was done to test both constructs ability to yield single proteins, of the correct size, that were recognized by the HA-antibody. Therefore, these constructs can be further tested for suitability in the analysis of Complex I assembly