Purification of Recombinant Calbindin D9k

Abstract

The calcium-binding protein calbindin D_9k is a relatively small member (8.5 kDa) of the EF-hand helix-loop-helix family of calcium-binding proteins. Unlike many other calcium-regulatory proteins in this family, binding of the two calcium ions to calbindin does not cause a large conformational change, which exposes a hydrophobic surface. Therefore, calbindin D_9K probably acts as a calcium buffer, rather than a calcium-trigger protein. Because there is no calcium-dependent conformational change, hydrophobic interactions as a purification step for recombinant calbindin is not feasible. The purification scheme described here for recombinant calbindin is based on the same procedure as used for purification of calbindin D_9k from intestine (1,2). The method starts with a heat shock step that takes advantage of calbindin’s unique stability. The second step is anion-exchange chromatography in the presence of ethylenediaminetetracetic acid (EDTA). The third step, gelfiltration, is used to remove small proteins, peptides, and other Escherichia coli components that do not elute from the ion exchange column with any distinct pattern. The fourth step is anion-exchange chromatography in the presence of calcium. Because calbindin changes its net charge upon binding calcium, it will elute at a different salt concentration in calcium compared to EDTA. Therefore, proteins that copurify in the EDTA step will be removed in the calcium step. The last anionexchange step will also remove any deamidated calbindin that may arise during the purification (3).