22] Affinity purification of recombinant proteins fused to calmodulin or to calmodulin-binding peptides

Abstract

The interaction between calmodulin (CaM) and its various ligands has been exploited for the affinity purification of recombinant proteins from Escherichia coli extracts in a number of systems. CaM is a calcium-binding protein that plays a central role in regulating a wide range of calcium dependent intracellular processes. This chapter focuses on the purification of fusion proteins containing the 26 amino acid calmodulin-binding peptide (CBP) derived from the C terminus of skeletal muscle myosin light-chain kinase using a solid support resin containing covalently bound calmodulin (CaM). Several alternative systems in which CaM is employed as a fusion partner are also described in the chapter. The high degree of specificity of CBP for CaM together with the stringent wash conditions, allowed because of the strength of the CBP-CaM interaction, results in the consistent recovery of highly purified CBP fusions. The small size of the peptide (<4 kDa) allows the recovery of soluble proteins of greater size than is attainable with the the currently popular protein tags. In addition, the CBP is less likely to affect the biological function of the protein of interest compared with the larger tags. It has been found that the CBP is translated efficiently in E. coli, and thus a wide range of proteins are consistently expressed to high levels when the CBP is positioned at the N terminus of the fusion.