Cloning and Expression of EFhd1 and EFhd2 Proteins

Mouse EFhd1 ΔNTD (residues 69−240) and the human EFhd2 core domain (residues 70−184) were amplified from full-length mouse EFhd1 (residues 1−240) and human EFhd2 (residues 1−240), respectively, using polymerase chain reaction (PCR). The amplified EFhd1 ΔNTD was cloned into a modified pET28a vector (Novagen) containing an N-terminal His₆ tag and a tobacco etch virus (TEV) protease cleavage site (Glu–Asn–Leu–Tyr–Phe–Gln/Gly). The amplified EFhd2 core domain was cloned into a modified pET41a vector containing gluta­thione S-transferase (GST) with a TEV protease cleavage site. Full-length EFhd1 was cloned into a modified pET28a vector (Novagen) with an N-terminal His₆-TEV tag. Full-length EFhd2 was cloned into a modified pET28a vector carrying an N-terminal His₆ tag.