Cloning and Expression of EhADH Protein

A DNA fragment of 2061 bp encoding the full-length of E. histolytica ehadh gene, was PCR amplified from the pExEhNeo-Ehadh112 plasmid (Bañuelos et al., 2005 (link)) using the oligonucleotides described in Table 1. The ehadh gene was cloned into the pGEX6P1 and pcDNA3 plasmids (Invitrogen) between BamHI and Xho1, or KpnI and BamH1 digestion sites, respectively. Escherichia coli C43 (DE3) and -DH5α bacteria (Invitrogen) were transformed with pGEX6P1-ehadh and pcDNA3-ehadh, respectively. Plasmids were purified by an affinity column (Qiagen) and automatically sequenced to corroborate the ehadh gene sequence.
To produce an EhADH recombinant protein (rEhADH), E. coli C43 (DE3) bacteria were transformed with the pGEX6P1-ehadh plasmid. The recombinant protein was induced with 1 mM IPTG and purified as described (Bañuelos et al., 2012 (link)).